Isolation, structural elucidation and synthesis of novel antineoplastic substances denominated &#34;combretastatins&#34;

ABSTRACT

New antineoplastic substances have been isolated, structurally elucidated and synthesized having a general structural formula of: ##STR1## wherein: R 1  is OH or OCH 3  ; 
     R 2  is H or OCH 3  ; or R 1  R 2  is --OCH 2  O--; 
     R 3  is H or OH; 
     R 4  is OH or OCH 3 . 
     These substances have been denominated &#34;combretastatin A-1, -A-2, -A-3, -B-1, -B-2, -B-3 and -B-4&#34;. Pharmaceutical preparation containing the substances and methods of treating a host inflicted with a neoplastic growth with the preparation is described.

This is a continuation of application Ser. No. 07/000,590, filed Jan. 6,1987, now abandoned.

INTRODUCTION

This invention relates to isolation, structural elucidation, andsynthesis of new antineoplastic substances herein denominated"combretastastin A-1", "combretastatin A-2", "combretastatin A-3","combretastatin B-4", said substance having a general structural formulaof: ##STR2## wherein: R₁ is OH or OCH₃ ;

R₂ is H or OCH₃ ; or R₁ R₂ is --OCH₂ O--;

R₃ is H or OH; and

R₄ is OH or OCH₃.

A portion of the work reported herein was funded under GrantCA-30311-01A3 from the National Cancer Institute and ContractN01-CM-97262 of the National Institute of Health.

BACKGROUND OF THE INVENTION

Tropical and subtropical shrubs and trees of the Combretaceae familyrepresent a practically unexplored reservoir of new substances withpotentially useful biological properties. Illustrative is the genusCombretum with 25 species (10% of the total) known in the primitivemedical practices of Africa and India for uses as diverse as treatingleprosy (See: Watt, J. M. et al, "The Medicinal and Poisonous Plants ofSouthern and Eastern Africa", E. & S. Livingstone, Ltd., London, 1962,p. 194) (Combretum sp. root) and cancer (Combretum latifolium). But onlya few species principally Combretum micranthum (used in northernZimbabwe for mental illness) (See: Ogan, A. U., Planta Medica, 1972, 21,210; and Malcolm, S. A. et al, Lloydia, 1969, 32, 512.) and C. zeyheri(for scorpion invenomation) (See: Mwauluka, K. et al, Biochem. Physiol.Pflanzen, 1975, 168, 15) have received any scientific study.

The present investigation was undertaken to determine the murine P388lymphocytic leukemia (PS system) inhibitory constituents of Combretumcaffrum (Eckl. and Zeyh) Kuntze (also as C. salicifolium E. Mey), apotentially useful lead which came out of the U.S. National CancerInstitute's world-wide exploratory survey of plants. In South Africathis tree is known by the Zulu as Mdubu (used as a charm) and isotherwise known as bushveld willow, bushwillow and rooiblaar. The timberis principally used on African farms as scrap wood and fuel.Interestingly, honey arising from the nectar of this tree is stronglybitter but no problems have been recorded from human consumption.

BRIEF SUMMARY OF THE INVENTION

New antineoplastic substances have been isolated, structurallyelucidated and synthesized which have a general structural formula of:##STR3## wherein: R₁ is OH or OCH₃ ;

R₂ is H or OCH₃ ; or R₁ R₂ is --OCH₂ O--;

R₃ is H Or OH: and

R₄ is OH or OCH₃ ;

The substances have been denominated combretastatins A-1, A-2, A-3, B-1,B-2, B-3 and B-4 and are specifically structured as follows, referencebeing made to the general structures shown above at I and II.

    ______________________________________                                        Combretastatin                                                                          Structure R.sub.1 R.sub.2                                                                              R.sub.3                                                                            R.sub.4                               ______________________________________                                        A-1       I         OCH.sub.3                                                                             OCH.sub.3                                                                            OH   OCH.sub.3                             A-2       I         --OCH.sub.2 O--                                                                            H    OCH.sub.3                               A-3       I         OH      OCH.sub.3                                                                            H    OCH.sub.3                             B-1       II        OCH.sub.3                                                                             OCH.sub.3                                                                            OH   OCH.sub.3                             B-2       II        OH      OCH.sub.3                                                                            H    OCH.sub.3                             B-3       II        OCH.sub.3                                                                             OCH.sub.3                                                                            H    OH                                    B-4       II        OCH.sub.3                                                                             H      H    OH                                    ______________________________________                                    

The substances are extracted from the stem wood of Combretum caffrum,with 1:1 methylene chloride and converted to a methylene chloridefraction that was partitioned between hexane and methanol-water followedby adjustment to 3:2 methanol-water and extract ion with methylenechloride. The methylene chloride fraction was separated by stericexclusion chromatography on Sephadex® LH-20 to obtain the fractions. Theisolation of the specific substances from the fraction is detailed inthe several examples reported below.

Accordingly, a principle object of the present invention is to isolateand elucidate the structure of new antineoplastic substances fromCombretum caffrum and to provide the methodology for the efficient andreliable replication thereof by synthetic procedures.

Another object of the present invention is to provide new and usefulpharmaceutical preparations containing one of the new antineoplasticsubstances as the essential active ingredient thereof.

These and still further objects as shall hereinafter appear arefulfilled by the present invention in a remarkably unexpected fashion aswill be readily discerned from a careful consideration of the followingdetailed description of preferred embodiments thereof especially whenread in conjunction with the several examples appended thereto.

DESCRIPTION OF PREFERRED EMBODIMENTS

In the course of the work which culminated in the present invention, weisolated a new substance that caused pronounced astrocyte reversal inthe NCI astrocytoma bioassay and was found to beR(-)-1-(3,4,5-tri-methoxy phenyl)-2-(3'-hydroxy,4'-methoxyphenyl)-ethanol. We named this substance "combretastatin".Meanwhile, attention was focused on uncovering the principal PS in vivoactive constituent(s) of Combretum caffrum fractions wherecombretastatin was not responsible for the biological activity. Theproblem was complicated by eventual loss of in vivo activity infractions obtained from the original large scale extraction of the stoneand fruit parts which led to combretastatin, Consequently the largeseals (77 kg) extraction of Combretum caffrum was repeated using thestem-wood and methylene chloride-methanol as solvent and the effortsresulted in the isolation, structural elucidation and ultimate synthesisof new substances forming the crux of this invention which, as willhereinafter appear, have a general structural formula determined to beeither: ##STR4## wherein: R₁ is OH or OCH₃ ;

R₂ is H or OCH₃ ; or R₁ R₂ is --OCH₂ O--;

R₃ is H or OH; and

R₄ is OH or OCH₃.

The methylene chloride fraction obtained by diluting the methylenechloride-methanol extract with water was subjected to solvent partitionbetween methanol-water (9:1-3:2) with hexane-methylene chloride. By thismeans the PS in vivo activity 38-41% life extension at 25-50 mg/kg andEd₅₀ 0.21 μg/mL was concentrated in the methylene chloride fraction.Steric exclusion chromatography of the active methylene chloridefraction in methanol on Sephadex LH-20 led to a fraction (30.6 g)preserving the PS in vivo activity. At this stage it was found mosteffective to proceed by partition chromatography on Sephadex LH-20 with3:1:1 hexane-toluene-methanol as mobil phase. The PS activity (30-48%life extension at 12.5-50 mg/kg)was nicely concentrated in an activefraction that was further purified by silica gel column chromatographyand eluted with hexane-ethyl acetate (3:1). After recrystallization ofthis active component herein named "combretastatin A-1" (0.70 g)obtained in 9.1×10⁻⁴ % yield it was unequivocally assigned the structureshown above as structure I when R₁ ═R₂ ═R₄ ═OCH₃ and R₃ is OH. (NSC600032, PS 26-29% increase in life extension at 2.75-11 mg/kg doselevels and Ed₅₀ 0.99 μg/mL, experiments at higher dose levels are now inprogress) and the companion cell growth inhibitory substance wasdenominated combretastatin B-1 and assigned structure shown as structureII above when R₁ ═R₂ ═R₄ ═OCH₃ and R₃ is OH (PS ED₅₀ 1.7 μg/mL, NSC601291), as follows.

Both tile ultraviolet and infrared spectra of combretastatins A-1 andB-1 suggested aromatic systems and this was further supported by thehigh resolution electron impact mass spectrum and assigned molecularformula C₁₈ H₂₀ O₆ and C₁₈ H₂₂ O₆ respectively. The 400 MHz ¹ H-NMRspectrum exhibited signals for four methoxy group protons and in generalindicated that combretastatin B-1 was a dihydro derivative of the A-1.Thus, further structural efforts were concentrated on determining thestructure of combretastatin A-1.

The 400 MHz ¹ H-NMR spectrum of combretastatin A-1 exhibited twomagnetically identical and relatively shielded aromatic protons atδ6.460, two AB spin systems totalling four aromatic protons with one ofthese appearing as a doublet at δ6.310 (J=8.64 Hz) and its counterpartat δ6.691 typical of two ortho coupled aromatic protons. The other ABspin system showed doublets at δ6.453 and 6.523 (J=12.2 Hz each). Atwo-proton signal at δ5.438 was readily exchanged for deuterium uponadding deuterium oxide suggesting the presence of phenolic groups andthat observation was confirmed by acetylating.

The mass spectrum of combretastatin B-1 gave a relatively smallmolecular ion at m/z 334 and two major fragment ions at m/z 181 (C₁₀ H₁₃O₃) and 153 (C₈ H₉ O₃) resulting from cleavage of the benzyl bond.Results of the mass spectral analysis suggested the presence of threemethoxyl groups in one aromatic ring and a methoxy and two hydroxygroups in the other aromatic ring. The relationship to combretastatinA-1 was easily established by selective catalytic hydrogenation of theA-1 to B-1. With the relationship of combretastatin A-1 to B-1established, examination of the ¹ H-NMR spectrum of combretastatin B-1was very helpful and revealed absence of the two proton doublets atδ6.453 and 6.523. With the relatively large coupling constant andintroduction of a 4-proton multiplet at δ2.851 typical of the benzylprotons of a bibenzyl (dihydrostilbene), combretastatin A-1 was assumedto be a stilbene.

Interpretation of the C¹³ -NMR spectrum (Table III) of combretastatinsA-1 and B-1 suggested each contained a 3,4,5-trimethoxy phenyl ring onthe basis of chemical shift additive rules. In the other aromatic ringthe position of the two carbons with proton substituents was readilyestablished, but the hydroxy vs. methoxy substituent arrangement wasambiguous. Eventually the substitution pattern in both aromatic ringswas established as shown for combretastatin A-1 and B-1 by applicationof nuclear Overhauser effect difference spectroscopy (NOEDS) methods.The most important observation here resulted from irradiation of themethoxy group at δ3.770 resulting in a 4.3% enhancement of thering-proton doublet at δ6.310.

The remaining uncertainty in completely assigning the structure ofcombretastatin A-1 on the basis of spectral evidence resided with thebridging olefin proton coupling constant J-12.2 Hz. Such couplingconstants fall in the range of 6-12 Hz for cis protons and 12-18 Hz fortrans protons with 10 and 17 Hz being typical values. While phenolicplant constituents of the stilbene type are generally isolated as thetrans-isomers (such as from Eucalyptus species) wood of the emeticSchotis brachypetala Sond (Leguminosae) has been shown to contain apentahydroxy cis,-stilbene. More recently Rheum rhatonticum L.(Polygonaceae) the commercial rhubarb has been found to contain fivecis-stilbenes and fourteen of the trans isomers. In the Rheum stilbenestudy a comparison of otherwise identical cis- and trans-stilbenes waspossible and the cis-olefin proton coupling constants were found to be12 Hz and the trans 16 Hz. These values correspond well with those laterrecorded in this investigation as a result of the total synthesessummarized in the sequel. Before this information became available forinterpreting significance of the combretastatin A-1 coupling constant at12.2 Hz the structure was unequivocally established by an x-ray-crystalstructure elucidation.

The crystal structure of combretastatin A-1 was solved by direct methodsusing a SHELX-84 computer technique combined with refinement anddifference syntheses based on SHELX-76. The molecular parameters wereestablished using the program PARST and the molecular representationshown below as FIG. 1 using PLUTO. ##STR5##

Combretastatin A-1 was obtained as plates in the moloclinic crystalsystem with space group P.2.1c. Bond lengths and angles were found to bethe expected order of magnitude. The cis-olefin geometry was confirmedby the torsion angle C(1)- C(1a)-C(1'a)-C(1') at -6(1)°. Normals to theleast-squares planes of the two phenyl rings were found inclined at66.3(2)° to each other and this distortion from an overall planarity ofthe molecule was further evidenced by the deviation from zero of thethree torsion angles C(6')-C(1')-C (1'a)-C(1a) at -16(1)°, C(1')C(1'a)-C(1a)-C(1) at -6(1)° and C(1'a)-C(1a)-C(1)-C(6) at -58(1)°. Mostlikely this results from the strong steric interaction in a singlemolecule between C(1) . . . C(6') of 3.372(8) A and C(6) . . . C(6') of3.273(9) A. Close contacts between O (2'). . . O(4), 3.242(6) A, O(2').. .O(5), 2.924(6) A and O(3'). . .O(3), 3.211(6) A are indicative of anintermolecular hydrogen bonding network. With results of the crystalstructure analysis in hand, the spectral analyses were firm includingthe 2.6% NOE enhancement of the proton at C-6' following irradiation ofthe C-2 proton: a result consistent with Z-geometry. The stage was thenset for total synthesis.

In order to obtain larger quantities of combretastatin A-1 for furtherbiological evaluation an efficient synthesis was devised based oncondensing protected aldehyde with the ylide derived from phosphoniumsalt. The important intermediate benzaldehyde required development of animproved synthesis. A selection of approaches to prepare utilizing otheravailable starting material proved inefficient and instead 2,3,4trihydroxybenzaldehyde proved to be a most effective starting substance.Reaction of phenol with sodium borate in water was found to selectivelyform the 2,3-borate ester and this allowed specific methylation of the4-hydroxyl group by dimethylsulfate. Acid hydrolysis of the borate esterafforded dihydroxybenzaldehyde which was more suitably reprotected byconversion to the 2,3-tert-butyldimethylsilyl ether. Because of opiniondifferences in the earlier literature regarding melting points forbenzaldehyde, it seemed necessary to provide some additional evidencefor the structure. For this purpose benzaldehyde was acetylated and theresulting diacetate was subjected to NMR irradiation of the methoxysignal at δ3.927 and an NOEDS experiment led to 5.3% enhancement of theC-5 proton doublet at δ6.982 thereby confirming the methoxy group atposition 4.

The phosphonium bromide was readily prepared via 3,4,5-trimethoxybenzylalcohol and the corresponding ylide (prepared in tetrahydrofuran usingbutyl lithium) was allowed to react with benzaldehyde. The product was amixture of olefins and in 92.5% yield with a Z/E ratio of 9:1 by H¹ -NMRanalysis. The Z-isomer (75%) was isolated by recrystallization (fromethanol ). Complete recovery of the remaining Z-isomer and the E-isomeron a preparative scale, either as the silyl ether derivatives or as theparent phenols, proved difficult but was readily accomplished using thediacetate derivatives. So the mixture of Z/E silyl ethers was treatedwith tetrabutylammonium fluoride to cleave the silyl protecting groupsand the phenols were acetylated and separated by silica gelchromatography to provide combretastatin A-1 diacetate and itstranscounterpart diacetate. Cleavage of disilylether with tetrabutylammonium fluoride and deacetylation of diacetate with potassiumcarbonate in methanol afforded combretastatin A-1 identical with thenatural product.

The 9:1 Z/E isomer ratio resulting from the Wittig reaction betweenbenzaldehyde and the ylide corresponding to phosphonium bromide requirescomment. In the past it appeared that the oxaphosphetanes resulting fromreaction of triphenyl-phosphonium alkylids and aldehydes werethermodynamically more stable in the threo configuration when preparedin the presence of lithium salts. The threo oxaphosphetane would then beexpected to give predominantly the corresponding trans olefin. In a"salt-free" solution the oxaphosphetane was expected to have the erythroconfiguration leading to a cis olefin. However, recently Schlosser andSchaub (See: J. Am. Chem. Soc., 1982, 104, 5821) have shown that thestereochemical environment around the group contributed by the ylide isof prime importance. Under "salt-free" conditions using(triethyl-phosphonio)-ethylide in tetrahydrofuran reaction withaldehydes gave high yields of trans olefins. In the Wittig reactionemployed to prepare combretastatin A-1 the presence of lithium bromidewas obviously unimportant compared to formation of an erythrooxaphosphetane in the most stable configuration. The sterically largesilyl protecting groups probably enhance the configuration of erythroover the preferred threo. Since ¹ H-NMR analysis of the crude Wittigreaction product showed a Z/E ratio of 9:1, it appears likely thatconfiguration of the intermediate oxaphosphetane was locked in place bysteric effects and that little if any steric "stereochemical drift"occurred between oxaphosphetane formation and production of thecis-olefin. To evaluate the preceding hypothesis the course of theWittig reaction was studied using ³¹ P-NMR (61.99 MHz) and the results(cf. experimental) clearly showed that there was no detectable cis-transinterconversion.

Preliminary biological evaluation of olefins 1 and 2 against the PS cellline gave some interesting insights into structural requirements forcell growth inhibitory activity. Combretastatin A-1 d lacerate was foundto be three-fold less active at PS Ed₅₀ 2.7 μg/mL than the parentnatural product. The trans-isomer counter part was essentially inactivewith PS Ed₅₀ 12 μg/mL. The silyl ether derivatives and were alsoinactive against the PS cell line. Most importantly combretastatin A-1was found to have the remarkable property of completely inhibitingmicrotubulin assembly in vitro at concentrations less than 1.5 μmolar(See Table I). Indeed, combretastatin A-1 appears to be an inhibitor oftubulin polymerization more potent than combretastatin and the wellknown tubulin inhibitors colchicine and podophylotoxin.

The newly characterized Combretum caffrum natural productsCombretastatin A-1 and B-1 were evaluated for in vitro interactions withtubulin (Table I). They were compared to combretastatin and to threeadditional well-characterized antimitotic agents, namely, colchicine,podophyllotoxin and steganacin, all of which bind at a common site ontubulin.

As appears from Table I, Combretastatin A-1 was more active thancombretastatin B-1 in its interactions with tubulin which agrees withits greater antineoplastic activity. Both compounds were significantlymore potent than the previously described combretastatin. In microtubuleassembly (Table I, Experiment I; see also Example 20 hereof), equivalentinhibition was observed with 2 μM combretastatin A-I, 3 μMcombretastatin B-1, and 11 μM combretastatin. The inhibition of assemblywith combretastatins A-1 and B-1 was comparable to that observed withpodophyllotoxin and greater than that observed with colchicine andsteganacin.

Combretastatin, podophyllotoxin, steganacin and colchicine all appear tobind at the same site on tubulin, as the first three agents act ascompetitive inhibitors of the binding of radiolabeled colchicine to theprotein. Combretastatins A-1 and B-1 were particularly potent asinhibitors of the binding of (³ H)colchicine to tubulin (Table I,Experiment II), significantly exceeding the inhibition observed withsteganacin, combretastatin and even, in the case of combretastatin A-1,podophyllotoxin.

                  TABLE 1                                                         ______________________________________                                        INHIBITION OF MICROTUBULE ASSEMBLY AND                                        BINDING OF COLCHICINE TO TUBULIN BY                                           COMBRETASTATIN A-1 AND COMBRETASTATIN B-1                                                 EXPERIMENT I  EXPERIMENT II                                                   MICROTUBULE   COLCHICINE                                                      ASSEMBLY      BINDING                                             DRUG        ID.sub.50 *(μM)                                                                          % of control                                        ______________________________________                                        Combretastatin A-1                                                                        2             2.2                                                 Combretastatin B-1                                                                        3             13                                                  Combretastatin                                                                            11            34                                                  Podophyllotoxin                                                                           3             13                                                  Steganacin  6             49                                                  Colchicine  6             --                                                  ______________________________________                                         *Defined as the drug concentration inhibiting the extent of microtubule       assembly by 50%.                                                         

Experimental Section

Synthetic intermediates were employed as received from Sigma-Aldrich.Solvents used for chromatographic procedures were redistilled. TheSephadex LH-20 (particle size 25-100 μm) used for steric exclusionchromatography was obtained from Pharmacia Fine Chemicals AB (Uppsala,Sweden). Silica gel 60 (70-230 mesh) utilized for column adsorptionchromatography and the Lobar silica gel 60 columns (size B) weresupplied by E. Merck (Darmstadt, Germany). Silica gel GHLF Uniplates(0.25 mm layer thickness) were obtained from Analtech, Inc., (Newark,Del.). The TL chromatograms were developed with anisaldehyde-acetic acidor eerie sulfate-sulfuric acid spray reagents (by heating atapproximately 150° C. for 5-10 min) or by application of ultravioletlight.

While the experimental procedures described above are directed to thepreparation of combretastatin A-1 and its conversion to combretastatinB-1, it should be realized that the approach described is equallyapplicable to the preparation of those combretastatins denominated A-2,A-3 and B-2, B-3 and B-4 as will become more apparent from a carefulconsideration of the Examples set forth below.

Thus, in each of the synthetic procedures, solvent extracts of aqueoussolutions were dried over anhydrous sodium sulfate. Ether refers todiethyl ether. Each pure specimen was colorless. The mutual identity ofnatural and synthetic specimens was established by comparison of infrared (NaCl) plates and ¹ H-NMR spectra combined with results from thinlayer chromatographic (TLC) comparisons in several solvents. All meltingpoints are uncorrected and were observed with a Kofler-type hot-stageapparatus. Ultraviolet spectra were obtained and recorded using aHewlett-Packard model 8540A UV/VIS spectro-photometer. Infra red spectrawere measured with a Nickolet FT-IR model MX-1 unit. Nuclear magneticresonance spectra were obtained with a Bruker WH-90 and AM-400instrument with deuteriochloroform as solvent and tetramethylsilane asthe internal standard. The chemical shifts were recorded using the δscale. The SFORD technique was used for determining multiplicities in ¹³C-NMR spectra. Nuclear Overhauser effect difference spectroscopy NOEDSexperiments were performed with a deuteriochloroform solution degassedsix times by the freeze-thaw technique. Mass spectral determinationswere made with a MS-50 instrument at the NSF Regional Facility,University of Nebraska, Lincoln, Nebr. Elemental microanalyses wereperformed at Mic-Anal, Tucson, Ariz. The X-ray crystal structuredetermination was performed with an Enraf-Nonius CAD-4 diffractometerand all computations were performed using a Sperry 1100 computer.

Isolation of Combretastatin A-2, A-3, and B-2

The stem wood (77 kg dry wt.) of Combretum caffrum was extracted with1:1 methylene chloride and converted to a methylene chloride fractionthat was partitioned between hexane and methanol-water (9:1) followed byadjustment to 3:2 methanol-water and extraction with methylene chloride.The methylene chloride fraction (827.9 g) from time solvent partitioningsequence was separated by steric exclusion chromatography on SephadexLH-20 to obtain fractions A and B.

Fraction A (28.6 g) was further separated on a column of Sephadex LH -20(2.5 kg) by partition chromatography employing hexane-toluene-methanol(3:1:1) to furnish an active fraction (2.07 G, PS Ed₅₀ 1.8×10⁻² μg/ml)which was dissolved in hexane-ethyl acetate (1:1, 5 ml) andchromatographed on a column (60×2.5 cm) of silica gel (60 g). Gradientelution from 4:1→1:1 hexane-ethyl acetate afforded in a 3:1 fraction thenext PS (0.7 g, Ed₅₀ 1.0×10⁻² μg/ml) active material. Rechromatographyin acetone (2 ml) over a long column (100×1.2 cm) of silica gel (45 g)using the gradient hexane-ethyl acetate 9:1→4:1 yielded in a 4:1fraction a pure specimen of combretastatin A-2 (0. 442 g, 5.74×10⁻⁴ %base on the dried plant, PS Ed₅₀ 2.7×10⁻² μg/ml) as a viscous oil: Rf.0.46 (1:1 hexane-ethyl acetate), UV (CH₃ OH)λmax(ε) 223 (17175), 303(7190); IR υ_(max) (NaCl) 3490, 1508, 1452, 1440, 1427, 1272, 1196,1129, 1114, 1085, 1042, 930 cm⁻¹ ; ¹ H-NMR (400 MHz) 3.750 (3H, s,OCH₃); 3.870 (3H, s, OCH₃), 5.520 (1H, s, OH, disappeared upon adding D₂O), 5.935 (2H,s,--OCH₂ O--), 6.383 (1H, d, J=12.16 Hz, --CH═CH--), 6.420(1H, d, J=12.16 Hz, --CH═CH --), 6.458 (1H, d, J=1.32 Hz, H-2 or H-6),6.483 (1H, d, J=1.32 Hz, H-6 or H-2), 6.731 (1H, d, J=8.4 Hz, H-5'),6.773 (1H, dd, J=8.4, 2.0 Hz, H-6'), 6.875 (1H, d, J=2.0 Hz, H-2'); ¹³C-NMR (see Table II); and HREIMS (m/z) 300.1001 (100, M⁺ calcd for C₁₇H₁₆ O₅, 300.0998), 285.0767 (4, C₁₆ H₁₃ O₅), 267.0666 (10, C₁₆ H₁₁ O₄),239.0714 (17,C₁₅ H₁₁ O₃).

Active fractions B (30.6 g) was also separated inhexane-toulene-methanol (3:1:1) by partition chromatography on SephadexLH-20 (2.5 kg). The PS active components were concentrated in twofractions: 8.11 g (PS, Ed₅₀ 2.7 μg/ml) and 1.57 g (PS Ed₅₀ 0.36 μg/ml).The latter fraction (1.57 g) was chromatographed on a column (70×2.5 cm)of silica gel (70 g) and eluted with hexane-ethyl acetate (4:1→41:1). Afraction eluted with 4:1 hexane-ethyl acetate gave a pure specimen ofcombretastatin A-3 (480.7 mg, 6.24×10⁻⁴ % yield, PS Ed₅₀ 2.6×10⁻² μg/ml)as a viscous oil: Rf. 0.40 (1:1, hexane-ethyl acetate) UV (CH₃ OH)λ_(max) (ε) 251(8090), 295(8895). UV(CH₃ OH+NaOCH₃) λ_(max) 259 (9078),279 (7383)), 296 (7402); IR υ_(max) (NaCl) 3430, 1583, 1509, 1458, 1441,1430, 1274, 1234, 1201, 1114, 1104 cm⁻¹ ; ¹ H-NMR (400 MHz) 3.668 (3H,s, OCH₃), 3.867 (3H, s, OCH₃), 3.886 (3H, s, OCH₃), 5.514, 5.680 (1H,each, brs, OH, D₂ O exchanged), 6.381 (1H, d, J=12.2 Hz, --CH═CH--),6.427 (1H, d, J=1.72 Hz, H-6), 6.439 (1H, d, J=12.2 Hz, --CH═CH--),6.535 (1H, d, J=1.72 Hz, H-2), 6.72 (1H, d, J=8.4 Hz, H-5'), 6.792 (1H,dd, J=8.4 Hz, 2.0 Hz, H-6'), 6.897 (1H, d, J=2.0 H_(z), H-2'), ¹³ C-NMR(refers to Table II); and HREIMS (m/z) 302.1156 ( 100, M⁺ calcd for C₁₇H₁₈ O₅ : 302.1154), 287.0919 (14, C₁₆ H₁₅ O₅), 269.0813 (14, C₁₆ H₁₃O₄).

The active fraction weighing 8.11 g was chromatographed in ethyl acetate(20 ml) on a column of silica gel (200 g). Elution with hexane-ethylacetate (3:1) and combination of earlier fractions furnished a 0.181 gfraction (PS, Ed₅₀ 0.19 μg/ml) that was further purified on a WhatmannHPLC column (500×10 mm) packed with partisil (M-9). Elution withhexane-2-propanol (9:1) at a flow rate of 0.56 ml/min afforded purecombretastatin B-2 (51.7 mg, 6.71×10⁻⁵ % yield, PS, Ed₅₀ 0.32 μg/ml asanother viscous oil: Rf. 0.42 (1:1 hexane-ethyl acetate); UV (CH₃ OH)λ_(max) (υ) 220 (22902), 280 (6120); IR υ_(max) (NaCl) 3437, 1595, 1512,1461, 1442, 1430, 1351, 1278, 1237, 1150 cm⁻¹ ; ¹ H-NMR (400 MH_(z))2.785 (1H, brs, --CH₂ --CH₂ --), 3.819 (3H, s, OCH₃), 3.856 (3H, s,OCH₃), 5.605, 5.748 (1H each, brs, OH, D₂ O exchanged), 6.264 (1H, d,J=1.88 Hz, H-6), 6.465 (1H, d, J=1.88 Hz, H-2), J=1.88 Hz, H-2), 6.648(1H, dd, J=8.12, 2.0 Hz, H-6'), 6.760 (1H, d, J=8.12 Hz, H-5'), 6.794(1H, d, J=2.0 Hz, H-2'); ¹³ C-NMR (see table 1); and HREIMS (m/z);304.1326 (30, M⁺ calcd for C₁₇ H₂₀ O₅ 304.1311), 167.0708 (100, C₉ H₁₁O₃), 137.0604 (65, C₈ H₉ O₂).

Isolation of Combretastins B-3 and B-4

The Combretum caffrum fraction reported earlier to containcombretastatin A-1 was submitted to further separation on a Partisil M-9column by HPLC with 9:1 hexane-2-propanol as solvent at a flow rate of 1ml/min to furnish 12.0 mg of combretastatin B-3 as a powder fromethanol-ether, mp 113°-15° C.: PS Ed₅₀ 0.4 μg/ml; UV (CH₃ OH) λ_(max)241 (ε8450), 281 (6907); UV (CH₃ OH+NaOCH₃) λ_(max) 246 (10190), 293(5699); IR υ_(max) 3400, 1590, 1507, 1457, 1420, 1240, 1126 cm⁻¹ ; ¹H-NMR (400 MHz), 2.796 (4H, s, --CH₂ --CH₂ --), 3.817 (6H, s, 2×OCH₃),3.829 (3H, s, OCH₃), 5.240, 5.350 (1H, each, brs, OH, D₂ Oexchangeable), 6.355 (2H, s, H-2, 6), 6.610 (1H, dd, J=7.88, 1.88 Hz,H-6'), 6.684 (1H, d, J=1.88 Hz, H-2'), 6.777 (1H, d, J=7.88 Hz, H-5');¹³ C-NMR (see Table II); and HREIMS (m/z) 304.1308 (M.sup. + 15%, calcdfor C₁₇ H₂₀ O₅ : 304.1311), 181.0863 (100, calcd for C₁₀ H₁₃ O₃ :181.0865), 123.0449 (9, calcd for C₇ H₇ O₂ : 123.0446).

The mixture remaining from the original separation of2,7-dihydroxy-3,4,6-trimethoxy-9,10-dihydrophenanthrene was furtherseparated by a series of chromatographic procedures starting with aLobar-A silica gel column and 3:7:0.1 hexane-methylene chloride-methanolas solvent followed by separation on three Lobar-A columns in seriesusing 6:4:0.5 hexane-chloroform-acetone as eluent. Where necessary,preparative thin layer chromatography on silica gel with 99:1 methylenechloride-methanol affected final separation. By this means, 35.8 mg ofcombretastatin B-4 was obtained as a viscous oil with PS Ed₅₀ 1.7 μg/ml;UV (CH₃ OH) λ_(max) 221 (ε19188), 280 (4942); UV (CH₃ OH+NaOCH₃) λ_(max)221 (24803), 280 (4111), 290 (3810); IR √υ_(max) 3400, 1595, 1512, 1460,1444, 1430, 1350, 1277, 1203, 1148 cm⁻¹ ; ¹ H-NMR (400 MHz ) 2.786 (4H,s, --CH₂ --CH₂ --), 3.757 (6H, s, 2×OCH₃), 5.196 (2H, broad, 2×H, D₂ Oexchangeable), 6.307 (1H, dd, J=2.0 Hz each, H-4), 6.322 (2H, brd, J=2.0Hz, H-2, 6), 6. 608 (1H, brd, J=7.8 Hz, H-6'), 6.687 (1H, brs, H-2'),6.755 (1Hd, J=7.8 Hz, H-5'); ¹³ C-NMR (refer to Table II); and HREIMS(m/z) 274.1208 (M⁺ 34.5%, calcd for C₁₆ H₁₈ O₄ : 274.1205), 152.0822(29, calcd for C₉ H₁₂ O₂ H : 152.0837), 151,0746 (15, calcd for C₉ H₁₁O₂ : 151.0759), 123.0450 (100, calcd for C₇ H₇ O₂ : 123.0446).

In final separation of the combretastatins, the bibenzyl(3'-hydroxy-3,4',5-trimethoxy bibenzyl) was isolated and recrystallizedfrom acetone-hexane to afford small needles melting at 108° C.: PS Ed₅₀1.7 μg/ml; UV (CH₃ OH) λ_(max) 222 (25412), 280 (6854); IR υ_(max) 3485,1609, 1595, 1511, 1469, 1452, 1425, 1207, 1146 cm⁻¹ ; ¹ H-NMR (90 MHz)2.82 (4H, s, --CH₂ --CH₂ --), 3.77 (6H, s, 2×OCH₃), 3.86 (3H, s, OCH₃),5.57 (1H, brs, OH, D₂ O exchangeable), 6.33 (3H, brs, H-2, 4, 6), 6.64(1H, dd, J=8.14, 1.8 Hz, H-6'), 6.77 (1H, d, J=8.14 Hz, H-5') 6.80 (1H,d, J=1.8 Hz, H-2'); and HREIMS (m/z) 288.1364 (M⁺ 22%, calcd for C₁₇ H₂₀O₄ : 288.1362), 151.0756 (5%, calcd for C₉ H₁₁ O₂ : 151.0759), 137.0603(100, calcd for C₈ H₉ O₂ : 137.0603).

The original fraction bearing2-hydroxy-3,4,6,7-tetramethoxy-9,10-dihydrophenanthrene chromatographedon a column of silica gel in 3:1 hexane-ethyl acetate to isolate thebibenzyl (4'-hydroxy-3,5-trimethoxy bibenzyl) (1.15 g) as a viscous oil;UV (CH₃ OH) λ_(max) 236 (ε3555), 280 (3212), UV (CH₃ OH+NaOCH₃) λ_(max)247 (6433), 280 (2506), 294 (1795); IR υ_(max) 3417, 1607, 1596, 1514,1461, 1429, 1204, 1150, 1066, 850, 690 cm⁻¹ ; ¹ H-nmr (90 MHz) 2.85 (4H,s, --CH₂ --CH₂ --), 3.76 (6H, s, 2×OCH₃), 5.15 (1H, brs, OH, D₂ Oexchangeable), 6.32 (3H, s, H-2, 4,6), 6.74 (2H, d, J=8.4 Hz, H-3', 5'),7.05 (2H, d, J=8.4 Hz, H-2', 6'), no aromatic solvent shift was observedwhen the spectrum was obtained in a mixture of C₆ D₆ -CDCl₃ ; and HREIMS(m/z 258.1255 (M⁺ 25%, calcd for C₁₆ H₁₈ O₃ : 258.1256), 152.0831 (33,calcd for C₉ H₁₂ O₂ : 152.0837), 107.0495 (100, calcd for C₇ H₇ O:107.0497).

A companion fraction from isolation of the combretastatin B-2 wasrechromatographed in 4:1 hexane-ethyl acetate on a column of silica gel,one of the fractions thereby prepared was used for final separation in9:1 hexane-2-propanol by HPLC on a column of Partisil M-9 with a flowrate of 1 ml/min. The result was 10 mg of bibenzyl(4'-hydroxy-3,4,5-trimethoxy bibenzyl) that recrystallized as needlesfrom acetone-hexane; mp 110-12; PS Ed₅₀ 0.25 μg/ml; IR υ_(max) 3411,1612, 1591, 1514, 1457, 1420, 1328, 1236, 1125, 1098, 840, 750 cm⁻¹ ; ¹H-nmr (90 MHz) 2.82 (4H, s, --CH₂ --CH₂ --), 3.82 (9H, s, 3×OCH₃), 4.99(1H, brs, OH, D₂ O exchangeable), 6.35 (2H, s, H-2 6), 6.75 (2H, d,J=8.6 Hz, H-3', 5'), 7.04 (2H, d, J=8.6 Hz, H-2', 6'); and HREIMS (m/z)288.136 (M⁺ 17% calcd for C₁₇ H₂₀ O₄ : 288.1362), 181.0863 (100, calcdfor C₁₀ H₁₃ O₃ : 181.0865), 107.0499 (100, calcd for C₇ H₇ O: 107.0497).

                  TABLE II                                                        ______________________________________                                        COMBRETASTATIN A-2, A-3, B-2, B-3, AND B-4,                                   13C-NMR (100.6 MHz) - CHEMICAL SHIFTS (δ)                               ASSIGNMENTS RELATIVE TO TETRAMETHYLSILANE                                     IN DEUTRIOCHLOROFORM.                                                         Carbon  A-2       A-3     B-2     B-3   B-4                                   ______________________________________                                        1       131.83    133.33  138.80  137.62                                                                              144.24                                2       108.57    108.92  108.05  105.63                                                                              106.73                                3       143.40    145.87.sup.a                                                                          145.61.sup.a                                                                          153.08                                                                              160.77                                4       134.40    138.79  133.92  134.86                                                                              98.10                                 5       148.40    151.98  152.20  153.08                                                                              160.77                                6       103.04    105.06  104.67  105.63                                                                              106.77                                1a      129.23    129.59  37.14   38.42 138.26                                1a'     128.88    128.96  38.07   37.23 36.89                                 1'      130.66    130.66  135.23  134.86                                                                              135.02                                2'      115.03    115.16  114.81  115.32                                                                              115.41*                               3'      145.80.sup.a                                                                            145.25.sup.a                                                                          144.98.sup.a                                                                          141.70                                                                              141.65**                              4'      145.32.sup.a                                                                            149.05  149.16  143.53                                                                              143.48**                              5'      110.45    110.45  110.82  115.70                                                                              115.71*                               6'      121.04    121.14  119.82  120.92                                                                              120.91                                ______________________________________                                         A-2; 101.35 (OCH.sub.2 O), 56.38, 55.92 (OCH.sub.3).                          A3: 60.99 (OCH.sub.3 at C4), 55.92, 55.70 (OCH.sub.3).                        B2: 60.97 (OCH.sub.3 at C4), 56.90, 55.90 (OCH.sub.3).                        B3: 56.15 (OC.sub.3), 60.94 (OCH.sub.3).                                      B4: 55.31 (OCH.sub.3),                                                        a, *, **in vertical column may be interchanged.                          

The administration of the several combretastatins herein disclosed andtheir pharmacologically active physiologicaly compatible derivatives isuseful for treating animals or humans having a neoplastic disease, forexample, acute lymphocytic leukemia and the like using the acceptedprotocols of the National Cancer Institute.

The dosage administered will be dependent upon the identity of theneoplastic disease; the type of host involved, including its age, healthand weight; the kind of concurrent treatment, if any; and the frequencyof treatment and therapeutic ratio.

Illustratively, dosage levels of the administered active ingredientsare: intravenous, 0.1 to about 200 mg/kg; intramuscular, 1 to about 500mg/kg; orally, 5 to about 1000 mg/kg; intranasal instillation, 5 toabout 1000 mg/kg; and aerosol, 5 to about 1000 mg/kg of host bodyweight.

Expressed in terms of concentration, an active ingredient can be presentin the compositions of the present invention for localized use about thecurls, intranasally, pharyngolaryngeally, bronchially, broncholially,intravaginally, rectally, or ocularly in a concentration of from about0.01 to about 50% w/w of the composition; preferably about 1 to about20% w/w of the composition; and for parenteral use in a concentration offrom about 0.05 to about 50% w/v of the composition and preferably fromabout 5 to about 20% w/v.

The compositions of the present invention are preferably presented foradministration to humans and animals in unit dosage forms, such astablets, capsules, pills, powders, granules, suppositories, sterileparenteral solutions or suspensions, sterile non-parenteral solutions orsuspensions, and oral solutions or suspensions and the like, containingsuitable quantities of an active ingredient.

For oral administration either solid or fluid unit dosage forms can beprepared.

Powders are prepared quite simply by comminuting the active ingredientto a suitably fine size and mixing with a similarly comminuted diluent.The diluent can be an edible carbohydrate material such as lactose orstarch. Advantageously, a sweetening agent or sugar is present as wellas a flavoring oil.

Capsules are produced by preparing a powder mixture as hereinbeforedescribed and filling into formed gelatin sheaths. Advantageously, as anadjuvant to the filling operation, a lubricant such as a talc, magnesiumsterate, calcium stearate and the like is added to the powder mixturebefore the filling operation.

Soft gelatin capsules are prepared by machine encapsulation of a slurryof active ingredients with an acceptable vegetable oil, light liquidpetrolatum or other inert oil or triglyceride.

Tablets are made by preparing a powder mixture, granulating or slugging,adding a lubricant and pressing into tablets. The powder mixture isprepared by mixing un active ingredient, suitably comminuted, with adiluent or base such as starch, lactose, kaolin, dicalcium phosphate andthe like. The powder mixture can be granulated by wetting with a bindersuch as corn syrup, gelatin solution, methylcellulose solution or acaciamucilage and forcing through a screen. As an alternative to granulating,the powder mixture can be slugged, i.e., run through the tablet machineand the resulting imperfectly formed tablets broken into pieces (slugs).The slugs can be lubricated to prevent sticking to the tablet-formingdies by means of the addition of stearic acid, a stearic salt, talc ormineral oil. The lubricated mixture is then compressed into tablets.

Advantageously the tablet can be provided with a protective coatingconsisting of a sealing coat or enteric coat of shellac, a coating ofsugar and methylcellulose and polish coating of carnauba wax.

Fluid unit dosage forms for oral administration such as syrups, elixirsand suspensions can be prepared wherein each teaspoonful of compositioncontains a predetermined amount of active ingredient for administration.The water-soluble forms can be dissolved in an aqueous vehicle togetherwith sugar, flavoring agents and preservatives to form a syrup. Anelixir is prepared by using a hydroalcoholic vehicle with suitablesweeteners together with a flavoring agent. Suspensions can be preparedof the insoluble forms with a suitable vehicle with the aid of asuspending agent such as acacia, tragacanth, methylcellulose and thelike.

For parenteral administration, fluid unit dosage forms are preparedutilizing an active ingredient and a sterile vehicle, water beingpreferred. The active ingredient, depending on the form andconcentration used, can be either suspended or dissolved in the vehicle.In preparing solutions the water-soluble active ingredient can bedissolved in water for injection and filter sterilized before fillinginto a suitable vial or ampule and sealing. Advantageously, adjuvantssuch as a local anesthetic, preservative and buffering agents can bedissolved in the vehicle. Parenteral suspensions are prepared insubstantially the same manner except that an active ingredient issuspended in the vehicle instead of being dissolved and sterilizationcannot be accomplished by filtration. The active ingredient can besterilized by exposure to ethylene oxide before suspending in thesterile vehicle. Advantageously, a surfactant or wetting agent isincluded in the composition to facilitate uniform distribution of theactive ingredient.

In addition to oral and parenteral administration, the rectal andvaginal routes can be utilized. An active ingredient can be administeredby means of a suppository. A vehicle which has a melting point at aboutbody temperature or one that is readily soluble can be utilized. Forexample, cocoa butter and various polyethylene glycols (Carbowaxes) canserve as the vehicle.

For intranasal instillation, a fluid unit dosage form is preparedutilizing an active ingredient and a suitable pharmaceutical vehicle,preferrably P.F. water, a dry powder can be formulated when insufflationis the administration of choice.

For use as aerosols, the active ingredients can be packaged in apressurized aerosol container together with a gaseous or liquefiedpropellant, for example, dichlorodifluoromethane, carbon dioxide,nitrogen, propane, and the like, with the usual adjuvants such ascosolvents and wetting agents, as may be necessary or desirable. Theterm "unit dosage form" as used in the specification and claims refersto physic ally discrete units suitable as unitary dosages for human andanimal subjects, each unit containing a predetermined quantity of activematerial calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical diluent, carrier orvehicle. The specifications for the novel unit dosage forms of thisinvention are dictated by and are directly dependent on (a) the uniquecharacteristics of the active material and the particular therapeuticeffect to be achieved, and (b) the limitation inherent in the art ofcompounding such an active material for therapeutic use in humans, asdisclosed in this specification, these being features of the presentinvention. Examples of suitable unit dosage forms in accord with thisinvention are tablets, capsules, troches, suppositories, powder packets,wafers, cachets, teaspoonfuls, tablespoonfuls, dropperfuls, ampules,vials, segregated multiples of any of the foregoing, and other forms asherein described.

The combretastatin active ingredients to be employed as antineoplasticagents can be easily prepared in such unit dosage form with theemployment of pharmaceutical materials which themselves are available inthe art and can be prepared by established procedures. Illustrative ofthe preparation of the unit dosage forms, and not as a limitationthereof, are set forth in Example 43 supra.

To further assist in the understanding of the present invention thefollowing examples are presented to more clearly disclose the presentinvention and not by way of limitation.

EXAMPLE 1 Plant Taxonomy

Stem wood of the South African tree Combretum caffrum (Eckl. and Zeyh)Kuntze was collected and identified as part of the National CancerInstitute-U.S. Department of Agriculture research program directed byDrs. John D. Douros, Matthew I. Suffness and James A. Duke. The stemwood (B817373) employed in this study was obtained in 1979.

EXAMPLE 2 Extraction and Solvent Partition Procedures

The dry stem wood (77 kg) of Combretum caffrum was subdivided bychipping and extract ed with 1:1 methylene chloride-methanol (320liters) at ambient temperature for eleven days. The methylene chloridephase was separated by addition of water (25% by volume) and the plantextract ion was repeated with another 320 liters of methylenechloride-methanol 1:1 as just described. The combined methylene chloridephases were concentrated to a crude extract weighing 1.42 kg and showingPS in vivo life extension of 27% at 100 mg/kg and PS Ed₅₀ 5.1 μg/mL Asolution of the methylene chloride fraction was partitioned 5× betweenhexane (18 liters) and methanol-water (9:1, 18 liters). After separatingthe hexane phase the methanol-water was adjusted to a concentration of3:2 and extracted (5×) with methylene chloride (18 liters). The hexaneextract (602.3 g) proved PS in vivo inactive and marginally activeagainst the cell line with Ed₅₀ 2.4 μg/mL. The PS in vivo activity(38-41% life extension at 25-50 mg/kg) and major cell growth inhibition(Ed₅₀ 0.21 μg/mL) was concentrated in the methylene chloride fraction(827.9 g) from the solvent partitioning sequence.

EXAMPLE 3 Isolation of Combretastatin A-1

The methylene chloride fraction from the solvent partitioning sequencewas dissolved in methanol (7×500 mL) and further separated by stericexclusion chromatography on columns of Sephadex LH-20 (7×2.5 kg). The PSactive (41% life extension at 12.5 mg/kg and Ed₅₀ 0.18 μg/mL fraction(30.6 g) was further separated in hexane-toluene-methanol (3:1:1)solution by partition chromatography on Sephadex LH-20 (2.5 kg). Furtherconcentration of the active components was achieved by this importantseparation step that gave a fraction (8.11 g) with 30-40% life extensionat 12.5-50 mg/kg and Ed₅₀ 2.7 μg/mL in the bioassay. The 8.11 g activefraction was chromatographed in ethyl acetate (20 mL) on a column ofsilica gel (200 g). Elution with hexane-ethyl acetate (3:1) led to twoactive fractions weighing 0.64 g and 2.25 g. Recrystallization of the2.25 g fraction from hexane-chloroform afforded a pure specimen ofcombretastatin A-1 (0.70 g, 9.1×10 ⁻⁴ % yield based on the dried plant)as plates melting at 113°-15° C.: UV (CH₃ OH) λ_(max) 233, 255, 298 m μ(Σ7145, 7766 7848); UV (CH₃ OH+CH₃ ONa) λ_(max) 232, 255, 288, 397 m μ(ε7323, 7679, 7038, 1983); IR (film) 3482, 3426, 1580, 1507, 1480, 1463,1452, 1328, 1290, 1238, 1125, 1092, 1000, 915, 850 cm⁻¹ ; ¹ H-NMR (400MHz) 3.597 (6H, s, 2×OCH₃ -3,5), 3.760 (3H, s, OCH₃ -4), 3.770 (3H, s,OCH₃ 4'), 5.438 (2H, br s, disappeared upon D₂ O exchange 2XOH-2', 3'),6.310 (1H, d, J_(AB) =8.64 Hz, H-5'), 6.453 (1H, d, J_(A'B') =12.2 Hz,--CH═CH--), 6.460 (2H, s, H-2,6), 6.523 (1H, d, J_(B'A') =12.2 Hz,--CH═CH--), 6.691 (1H, d, J_(BA) =8.6 Hz, H-6'); ¹³ C-NMR (see TableIII); and HREIMS (m/z) 332.1248 (M⁺ 100%, calcd 332.1259 for C₁₈ H₂₀ O₆)and 317.1005 (M⁺ --CH₃ 93.7%, C₁₇ H₁₇ O₆). Anal. Calcd for C₁₈ H₂₀ O₆ :C, 65.05; H, 6.06. Found: C, 64.80; H, 6.08.

EXAMPLE 4 Isolation of Combretastatin B-1

The 0.6 g active fraction from the silica gel column chromatographproduced by Example 3 was rechromatographed using two Lobar B columns inseries. Elution with hexane-ethyl acetate (7:3) provided combretastatinB-1 as an oil (39.6 mg) in 5.1×10⁻⁵ % yield based on the dry plantstarting material. The colorless gummy combretastatin B-1 (3) exhibitedUV (CH₃ OH) λ_(max) 239, 270 m μ (ε5845, 1949); UV (CH₃ OH+CH₃ ONa)λ_(max) 240, 256 m μ (ε5860, 5949); IR (film) 3424, 3408, 1590, 1508,1457, 1288, 1126, 1093 cm⁻¹ ; ¹ H-NMR (400 MHz) 2.851 (4H, m, --CH₂--CH₂ --), 3.827 (3H, s, OCH₃ -4'), 3.831 (6H, s, 2×OCH₃ -3,5), 3.856(3H, s, OCH₃ -4), 5.382, 5.398 (1H each, D₂ O exchangeable, 2×OH-2',3'), 6.390 (1H, d, J_(AB) =8.36 Hz, H-5'), 6.420 (2H, s, H-2,6), 6.577(1H, d, J_(BA) =8.36 Hz, H-6'); ¹³ C-NMR (refer to Table III); andHREIMS (M/z), 334.1417 (27.2%, M⁺ calcd, C₁₈ H₂₂ O₆ for 334,1416,181.0861 (100, calcd C₁₀ H₁₃ O₃ for 181.0865) and 153.0549 (59.6 calcdC₈ H₉ O₃ for 153.0552).

                  TABLE III                                                       ______________________________________                                        Combretastatin A-1 and B-1 .sup.13 C-NMR (100 MHz)                            Chemical Shift Assignments relative to                                        Tetramethylsilane in Deuteriochloroform Solution                              Structure                                                                     Assign. No.     A-1      B-1                                                  ______________________________________                                        1               132.49*  138.18                                               2               106.13   105.67                                               3               152.80   153.05                                               4               132.67*  132.35                                               5               152.80   153.05                                               6               106.13   105.67                                               1a              130.21** 36.49**                                              1'a             124.06** 31.82**                                              1'              117.91   121.55                                               2'              141.72   142.19                                               3'              137.42   136.21                                               4'              146.37   145.40                                               5'              102.98   102.52                                               6'              120.17   120.32                                               3,5-OCH.sub.3   55.85    56.12                                                4-OCH.sub.3     60.79    60.18                                                4'-OCH.sub.3    56.16    56.18                                                ______________________________________                                         *, **Assignments may be interchanged.                                    

EXAMPLE 5 Acetylation of Combretastatin A-1

A solution of combretastatin A-1 (5 mg) in 0.5 mL of 1:1 aceticanhydride-pyridine was allowed to stand overnight at room temperature.The volatile components were evaporated under a stream of nitrogen andthe product crystallized from hexane-ethyl acetate to afford colorlessplates of the acetate: mp 133°-35°; IR (film) 1775, 1579, 1503, 1454,1420, 1206, 1174, 1127, 1088, 1010 cm⁻¹ ; ¹ H-NMR (400 MHz) 2.264, 2.299(3H each, s, COCH₃), 3.664 (6H, s, 2×OCH₃), 3.807 (3H, s, OCH₃) 3.813(3H, s, OCH₃), 6.361 (1H, d, J_(AB) =11.90 Hz, --CH═CH--), 6.442 (2H, s,H-2,6), 6.548 (1H, d, J_(BA) =11.90 Hz, --CH═CH--), 6.726 (1H, d,J_(A'B') =8.7 Hz, H-5'), 7.025 (1 H, d, J_(B'A') =8.7 Hz, H-6'); andHREIMS (m/z) 416.1463 (60M⁺ calcd C₂₂ H₂₄ O₈ for 416.1471), 374.1363(70. M+H)⁺ --COCH₃ C₂₀ H₂₂ O₇), and 332.1263 (100, (M+2H)⁺ -2×COCH₃ C₁₈H₁₈ O₆).

EXAMPLE 6 Combretastatin B-1 prepared by Hydrogenation of CombretastatinA-1

A mixture of combretastatin A-1 (35 mg) in methanol (15 mL) and 5% Pd/C(10 mg) was treated with a positive pressure of hydrogen at ambienttemperature overnight. Catalyst was removed by filtering the yellowsolution and the product was purified by preparative layerchromatography by Whatman KC18 plates with acetone-methylene chloride(1:11.5) as mobile phase. The product was identical (by TLC, IR and NMR)with natural combretastatin B-1.

EXAMPLE 7 The Crystal and Molecular Structure of Combretastatin A-1

Single crystals of combretastatin A-1 were obtained fromhexane-chloroform. The crystals were small very thin plates and as suchnot entirely suitable for X-ray analysis. However, one such crystal wasselected for irradiation. During the data collection, intensities ofthree standard reference reflections monitored every hour and centeringchecked every hundred measured relections. Intensities were correctedfor Lorentz and polarization effects but not for absorption. Thestructure was solved by direct methods using a preliminary version ofSHELX-84 which yielded in an E map, 23 of the 24 non-hydrogen atoms.Subsequent refinement and difference syntheses using SHELX-76 enabledlocation of the remaining non-hydrogen atom. Hydrogen atoms of thephenyl rings and the olefinic group were placed in calculated positionswith a single temperature factor. Methyl hydrogens were treated as rigidgroups with a single temperature factor. The two hydroxyl hydrogens wereinitially placed as located in a difference map and constrained to rideat 1.00 A from their parent oxygens. In the final refinements all atomswere treated with isotropic thermal motion. Molecular parameters wereobtained using PARST (See: Nardelli, M., Comput Chem 1983, 7, 95) and adrawing of the molecule using PLUTO (See: Motherwell,W. D. S., PLUTOPlotting Program, Cambridge University, England, 1974, unpublished).Further details of the data collection solution and refinement of thestructure are shown in Table IV, below. Final atomic coordinates of themolecule are shown in Table V, below, and a perspective view with atomicnomenclature is shown at FIG. 1, above. Relevant molecular parametersare reported in Table VI below.

                  TABLE IV                                                        ______________________________________                                        Crystallographic Data and Summary of Intensity                                Data Collection and Structure Refinement                                      for Combretastatin A-1                                                        ______________________________________                                        Molecular formula     C.sub.18 H.sub.20 O.sub.6                               Mr. g mol             332.35                                                  Crystal system        monoclinic                                              Space group            -p2.sub.1 / -c                                         T, K                  294                                                      -a, Å            10.497 (2)                                               -b, Å            6.717 (2)                                                -c, Å            22.746 (4)                                              β, °      96.11 (2)                                               V, A.sup.3            1594.7 (6)                                              Z                     4                                                        -d calc, g cm.sup.-3 1.38                                                    Crystal dimensions, mm                                                                              0.06 × 0.16 × 0.34                          Radiation wavelength MoKα, Å                                                              0.7107                                                  Crystal decay, %      1                                                       μ, cm.sup.-1       0.972                                                   F (000)               704                                                     Scan Mode             ω-2 θ                                       Scan width in ω, °                                                                     (0.64 + 0.35 tan 0)                                     Aperture width, mm    (1.12 + 1.05 tan 0)                                     Aperture length, mn   4                                                       Final acceptance limit                                                                              20 τ at 20° min                              Maximum recording time, s                                                                           40                                                      Scan range, 2 θ 2-46                                                    No. of reflections collected                                                                        1793                                                    No. of reflections observed                                                                         1265                                                    (with Irel > 2 τ Irel)                                                    No. of parameters     0.076                                                   R = ε||Fo |-| Fe                  ||/ε|Fo|                                                0.74                                                    Rw = εw.sup.1/2 ||Fo |-|Fe        ||/εw.sup.1/2 |Fo|                                      (τ.sup.2 F)-1                                       ______________________________________                                    

                  TABLE V                                                         ______________________________________                                        Fractional atomic coordinates (× 10.sup.4) and                          temperature factors (Å.sup.2 × 10.sup.3) for non-hydrogen           atoms                                                                         of combretastatin A-1                                                                x/a    y/b        z/c       Uiso                                       ______________________________________                                        C(1)     6517(6)  4976(9)    1646(3) 37(2)                                    C(2)     6985(6)  6387(10)   1286(3) 36(2)                                    C(3)     8212(6)  6159(9)    1116(3) 37(2)                                    O(3)     8750(4)  7487(7)    742(2)  50(1)                                    C(31)    7995(7)  9107(10)   512(3)  52(2)                                    C(4)     8988(6)  4633(9)    1319(3) 32(2)                                    O(4)     10241(4) 4564(6)    1183(2) 42(1)                                    C(41)    10442(7) 3270(11)   711(3)  57(2)                                    C(5)     8514(6)  3209(9)    1675(3) 33(2)                                    O(5)     9352(4)  1693(6)    1862(2) 39(1)                                    C(51)    8917(6)  157(10)    2230(3) 42(2)                                    C(6)     7286(5)  3361(10)   1845(3) 34(2)                                    C(1a)    5250(6)  5280(10)   1852(3) 42(2)                                    C(1'a)   4222(6)  4122(9)    1822(3) 38(2)                                    C(1')    3959(5)  2190(9)    1519(3) 34(2)                                    C(2')    2868(5)  1124(9)    1638(3) 34(2)                                    O(2')    2148(4)  1839(7)    2063(2) 45(1)                                    C(3')    2509(6)  -621(9)    1350(3) 34(2)                                    O(3')    1431(4)  -1586(7)   1510(2) 51(1)                                    C(4')    3198(5)  -1314(9)   916(3)  32(2)                                    O(4')    2702(4)  -3024(7)   635(2)  47(1)                                    C(41')   3459(7)  -3992(11)  238(3)  57(2)                                    C(5')    4285(6)  -324(9)    780(3)  36(2)                                    C(6')    4648(6)  1401(9)    1085(3) 38(2)                                    ______________________________________                                    

                  TABLE VI                                                        ______________________________________                                        Data Pertinent to the Molecular                                               Geometry and Packing of Combretastatin A-1                                    ______________________________________                                        Bond Lengths. A                                                               Ring C--C         in range 1.366(9)-1.401(9)                                  C(ring) --O       in range 1.375(8)-1.389(8)                                  O--C(methyl)      in range 1.413(8)-1.434(8)                                  Olefin C(1a)--C(1'a)       1.326(9)                                           C(1)--C(1a)                1.471(9)                                           C(1')--C(1'a)              1.482(9)                                           Bond Angles. °                                                         Ring C--C--C      in range 116.1(6)-122.7(6)                                  C(ring)--O--C--(methyl)                                                                         in range 114.4(5)-118.6(5)                                  C(ring)--C(ring)--O                                                                             in range 114.5(5)-124.9(5)                                  C(1)--C(1a)--C(1'a)        131.4(6)                                           C(1a)--C(1'a)--C(1')       130.4(6)                                           Torsion angles. °                                                      C((6')--C(1')--C(1'a)--C(1a)                                                                         -16(1)                                                 C(1')--C(1'a)--C(1a)--C(1)                                                                           -6(1)                                                  C(1'a)--C(1a)--C(1)--C(6)                                                                            -58(1)                                                 Equations of Planes                                                           C(1)--C(2)--C(3)--C(4)--C(5)--C(6)                                                                  -.272x - .528y -                                                              .805z = -6.51                                           C(1')--C(2')--C(3')--C(4')--C(5')--C(6')                                                            -.489x + .533y -                                                              .691z = -3.444                                          Non-bonded contacts, A                                                         (symmetry coordinate applied to second atom)                                 O(2') . . . O(4)                                                                             3.242(6)   x - 1, y, z                                         O(2') . . . O(5)                                                                             2.924(6)   x + 1, y, z                                         O(3') . . . O(3)                                                                             3.211(6)   x - 1, y - 1, z                                     ______________________________________                                    

EXAMPLE 8 Synthesis of 2,3-dihydroxy-4-methoxy-benzaldehyde

To a vigorously stirred solution of sodium borate-decahydrate (borax, 30g) in 600 mL of water was added 2,3,4-trihydroxy-bennzaldehyde (5 g,32.4 mmol). The yellow solution was stirred at room temperature for 30rain followed by dropwise and simultaneous addition (over 30 min) ofsodium hydroxide (4.0 g, 100 mmol) in water (50 mL) and dimethylsulfate(9.45 mL, 100 mmol). Vigorous stirring was continued overnight and conc.hydrochloric acid was added to pH 1. After stirring for an additional 30min the mixture was extracted with chloroform (5×300 mL). The organiclayer was once washed with brine, dried and evaporated to yield aslightly yellowish solid which on crystallization from ethylacetate-hexane afforded slightly yellowish colored needles (3.9 g, 72%),mp 116°-17° C.: (lit. 118°-119° C.), IR (film) 3374, 1646, 1505, 1461,1443, 1278, 1210, 1106, 636 cm⁻¹ ; ¹ H-NMR 3.987 (3H, s, OCH.sub. 3),5.466 (1H, brs, OH-3, D₂ O exchanged) 6.617 (1H, d, J_(AB) =8.62, H-5),7.147 (1H, d, J_(BA) =8.62 Hz, H-6), 9.757 (1H, s, CHO) 11.113 (1H, brs,OH-2, D₂ O exchanged); and HREIMS (m/z, 168.0419 (M⁺, 100%; calcd168.0423 for C₈ H₈ O₄).

EXAMPLE 9 Acetylation of 2,3-dihydroxy-4-methoxy-benzaldehyde

The diphenol prepared pursuant to Example 8 (100 mg) was acetylated withacetic anhydride-pyridine to afford the diacetate as crystals fromacetone-hexane: mp 126.5°-28.5° C. IR (film) 1772, 1693, 1609, 1506,1459, 1370, 1295, 1202, 1174, 1101 and 807 cm⁻¹ ; ¹ H NMR (400 MHz)2.331 (3H, s, COCH₃), 2.386 (3H, s, COCH₃), 3.927 (3H, s, OCH₃), 6.982(1H, d, J_(AB) =8.8 Hz, H-5), 7.749 (1H, d, J_(BA) =8.8 Hz, H-6), 9.907(1H, s, CHO); and HREIMS (m/z) 210.0524 (M⁺, 20%, calcd C₁₀ H₁₀ O₅ for210.0528 and 168.0417 (100% [M+H]⁺ COCH₃, C₈ H₈ O₄). Anal. calcd for C₁₂H₁₂ O₆, C, 57.15; H, 4.75. Found: C, 57.18; H, 4.75.

EXAMPLE 10 3,4,5-trimethoxy-benzyltriphenylphosphonium bromide

A solution of triphenylphosphine (4.2 g) in toluene (10 mL) was added toa stirred solution of 3,4,5-trimethoxybenzyl bromide (4.0 g) in toluene(15 mL) and stirring was continued for 24 hours. The phosphonium bromidethat separated (8.0 g, 99%) was collected and dried under vacuum, mp223-4 (lit 222°-23° C.).

EXAMPLE 112,3,-Bis-[(tert-butyldimethylsilyl)-oxy]-4-methoxy-benzaldehyde

Diisopropylethylamine (1.6 mL, 9.0 mmol) was added to a stirred solution(under argon) of 2,3-dihydroxy-4-methoxy-benzaldehyde (0.50 g, 2.98mmol) in dimethylformamide (5 mL) followed by tert-butyldimethylsilylchloride (1.0 g, 6.66 mmol). The reaction mixture was stirred at roomtemperature for 20 min. Ice (10 g) was added and the mixture wasextracted with ether (3×15 mL). The ethereal solution was washed withwater (15 mL), saturated sodium bicarbonate (2×10 mL), water (20 mL),and solvent evaporated to yield silyl ether as a chromatographicallyhomogeneous oil (1.15 g, quantitative) that crystallized from methanol:mp 74.5°-76° C.; IR (film) 2931, 1684, 1586, 1454, 1292, 1264, 1099,843, 827 cm⁻¹ ; ¹ H-HMR, 0.132 (12H, s, 4×SiCH₃), 0.987 (9H, s, 3×CH₃),1.038 (9H, s, 3×,CH₃), 1.038 (9H, s, 3×CH₃), 6.612 (1H, d, J_(AB) =8.7Hz, H-5), 7.483 (1H, d, J_(BA) =8.7 Hz, H-6), 10.225 (1H, s, CHO); andHREIMS (m/z) 381.1915 (5, M⁺ CH₃, calcd 381.1917 for C₁₉ H₃₃ O₄ Si₂),339.1429 (100, M⁺ C₄ H₉, calcd 339.14148 for C₁₆ H₂₇ O₄ Si₂). Anal.calcd for C₂₀ H₃₆ O₄ Si₂, C, 60.56;H, 9.15. Found; C, 60.38;H, 9.28.

EXAMPLE 12 2', 3'-Bis-[(tert-butyldimethylsilyl)-oxy-(Z) and(E)-Combretastatin A-1 Synthetic Procedure

Butyllithium (20 mL, 1.5M in hexane, 30 mmol) was added (under argon) toa suspension of 3,4,5-trimethoxybenzyl-triphenyl-phosphonium bromide(15.7 g, 30 mmol) in tetrahydrofuran (450 mL) at -15°. The resultingdeep reddish solution was allowed to stir at room temperature for 30min. Aldehyde (11.09 g, 28.0 mmol) was added and the reaction mixturewas diluted with ice-c old water and extracted with ether (3×250 mL).The ethereal solution was washed with water and solvent was evaporatedto yield a crude product which was crystallized from ethanol to affordpure Z-isomer (11.0 g) and a mixture (1:1, by ¹ H-NMR) of Z/E isomer(3.5 g, total yield 92.5%). The Z-isomer recrystallized frommethanol-ethyl acetate to furnish colorless needles: mp 117°-18° C.; IR(film) 1580, 1507, 1496, 1472, 1456, 1445, 1420, 1248, 1129, 1102, 1010,840, 780 cm⁻¹ ; ¹ H-NMR (400 MHz) 0.105 (6H, s, 2×Si-- CH₃), 0.190 (6H,s, 2×Si CH₃), 0.999 (9H, s, 3×CH₃), 1.038 (9H, s, 3×CH₃), 3.674 (6H, s,2×OCH₃), 3.738 (3H, s, OCH₃), 3.835 (3H, s, OCH₃), 6.358 (1H, d,J_(A'B') =12.0 Hz, --CH═CH--), 6.361 (1H, d, J_(AB) =8.7 Hz H-5'), 6.584(1H, d, J_(B'A') =12.4 Hz, --CH═CH--), 6.619 (2H, s, H-2, 6), 6.910 (1H,d, J_(BA) =8.7 Hz, H-6'); and HREIMS (m/z) 560.2941 (90%, m⁺, calcd560.2989 for C₃₀ H₄₈ O₆ Si₂), 488.2060 (100, M⁺ C₅ H₁₂, C.sub. 25 H₃₆ O₆Si₂). Anal. calcd for C₃₀ H₄₈ O₆ Si₂, C, 64.25;H, 8.63. Found: C,64.03;H, 8.70.

EXAMPLE 13 Purified E-isomer

A small portion of the Z/E mixture produced according to Example 12 waschromatographed on a silica gel column and eluted with hexane-ethylacetate (49:1). The fraction enriched with the E-isomer crystallizedfrom methanol-ethyl acetate to afford pure E-isomer as colorless platesmelting at 139°-40° C.: IR (film) 1581, 1507, 1496, 1472, 1463, 1456,1444, 1239, 1130, 1101, 840, 785 cm⁻¹ ; ¹ H-NMR (400 MHz) 0.114 (6H, s,2×SiCH₃), 0.133 (6H, s, 2×SiCH₃), 0.999 (9H, s, 3×CH₃), 1.092 (9H, s,3×CH₃), 3.793 (3H, s, OCH₃), 3.862 (3H, s, OCH₃), 3.884 (6H, s, 2×OCH₃),6.556 (1H, d, J_(AB) =8.72 Hz, H-5'), 6.716 (2H, s, H-2,6), 6.805 (1H,d, J_(A'B') =16.44 Hz, --CH=CH--), 7.198 (1H, d, J_(BA) =8.72 Hz, H-6'),7.308 (1H, d, J_(B'A') =16.44 Hz, --CH═CH--); and HREIMS (m/z) 560.3151(100, M Calcd C₃₀ H₄₈ O₆ Si₂ for 560.2989) 488.2059 (90, M⁺ C₅ H₁₂, C₂₅H₃₆ O₆ Si₂). Anal. Calcd for C₃₀ H₄₈ O₆ Si₂ 1/2 H₂ O: C, 63.23;H, 8.66.Found: C, 63.32;

EXAMPLE 14 Z and E Isomers

In another experiment, when 1.5 equivalents of n-butyllithium was usedper equivalent of phosphonium bromide, the ratio of Z/E isomer changeddramatically from 9:1 to 3.5:1.

EXAMPLE 15 ³¹ P-NMR Evaluation

Phosphonium bromide (0.523 g, 1.0 mmol) in dry tetrahydrofuran (20 mL)was treated (under argon) with 1.0 molar equivalent of n-butyllithium at-15° to generate the ylide. An aliquot (2.0 mL, 0.10 mmol) of the ylidesolution was transfered to an NMR tube (10 mm) and frozen (liquidnitrogen). A solution of aldehyde (5d, 39.0 mg, 0.098 mmol) intetrahydrofuran-d₈ (1 mL) was added and the frozen sample was warmed to-80° in the NMR probe. Examination of the spectrum (at -80° showed threesharp singlets at δ24.538 (ylide), 7.553 (cis oxaphosphetane) and δ8.0ppm (trans oxaphosphetane) integrating in the ratio 15:65:1respectively. On warming to 60° during 10 min the cis-oxaphosphetane wasformed at the expense of the ylide and the ratio changed to 9:69:1 Whiledisappearance of the ylide was still in progress a new broad singletstarted appearing (-50°, 10 min) at 28.4 ppmm, due to formation oftriphenylphosphine oxide (as a lithium bromide complex), the transoxaphosphetane signal disappeared and the ratio of signals fromdownfield to upfield was 11:3:64:0. The disappearance of cisoxaphosphetane and appearance of triphenylphosphine oxide was monitoredat -30° (10 minutes after -50°) and -10° (12 minutes after -30°) to givethe ratios 11:23 and 33:18 respectively. After another 12 minutes at 25°C. the oxaphosphetane disappeared completely. The results clearlyindicate that there was no interconversions of cis to transoxaphosphetanes. The 10% of E-isomer may have been formed due toisomerization during isolation. The shift of ³¹ P in thetriphenylphosphine oxide lithium bromide complex was found to changewith temperature as follows: -50° (28.4), -30° (28.0 used as referenceper Reitz et al, J. Am. Chem. Soc., 1984, 106, 1873), -10° (27.9), and25° (26.3).

EXAMPLE 16 2', 3'-diacetoxy-4'-methoxy-Z-combretastatin A-1

To a 1.8 g sample of the isomer mixture in tetrahydrofuran (10 mL) wasadded tetrabutylammonium fluoride 8 mL of a 1M solution intetrahydrofuran and the mixture was stirred (under argon) at roomtemperature for 15 minutes. Ethyl ether (50 mL) was added and thesolution was washed with water (2×50 mL) and the solvent removed underreduced pressure). The residue was acetylated in 4 mL of 1:1 aceticanhydride-pyridine. After stirring overnight the acetylation mixture waspoured into ice-water, extracted with ether (3×50 mL), washedsuccessively with 1N-hydrochloric acid (2×25 mL), saturated sodiumbicarbonate solution (2×25 mL) and water (50 mL). Removal of solventfurnished a gummy residue which was chromatographed on a column ofsilica gel (50 g). Gradient elution with hexane-ethyl acetate (9:1→1:1)afforded 0.55 g of Z-isomer, and 0.60 g of E-isomer. The Z-isomer wasrecrystallized from hexane-ethyl acetate to give colorless prisms, mp133°-35° identical and diacetate prepared from natural combretastatinA-1. Anal. calcd for C₂₂ H₂₄ O₈ : C, 63.46;H, 5.81. Found: C, 63.37;H,5.79.

EXAMPLE 17 2',3'-diacetoxy-4'-methoxy-E-combretastatin A-1

The E-isomer collected from Example 16 was recrystallized as needles mp172°-73° C. from hexane-ethyl acetate: IR (film) 1775, 1582, 1507, 1455,1295, 1206, 1173, 1126, 1089, 670 cm⁻¹. ¹ H-NMR (400 MHz) 2.313 (3H, s,COCH₃), 2.348 (3H, s,COCH₃), 3.863 (6H, s, 2×OCH₃), 3.897 (3H, s, OCH₃),3.899 (3H, s, OCH₃), 6.669 (2H, s, H-2, 6), 6,870 (1H, d, J_(AB) =16,02Hz, --CH═CH--), 6.897 (1H, d, J_(A'B') =8.5 Hz, H-5'), 6.917 (1H, d,J_(BA) =16.02 Hz --CH═CH--), 7.470 (1H, d, J_(B'A') =8.5 Hz, H-6') andHREIMS (m/z) 416.1486 (33, M⁺, calcd 416.1471 for C₂₂ H₂₄ O₈), 374,1347(39, M+H COCH₃), 332.1234 (46, M+2H⁺ -2×CH₃ CO). Anal. calcd for C₂₂ H₂₄O₈ 1/2 H₂ O, C, 62.11; H, 5.92. Found: C, 62.27;H, 5.73.

EXAMPLE 18 Combretastatin A-1

Method A. A 60 mg sample of the synthetic diacetate in methanol (3 mL)was stirred (under argon) with potassium carbonate (50 mg) for 1 hour.Hydrochloric acid (1N) was added and the phenol was extracted withchloroform (3×10 mL), washed with water (10 mL) and solvent removed. Theproduct was passed through a pipette of silica gel (1.0 g) to yieldcombretastatin A-1 (45 mg 94%). The viscous oil crystallized fromhexane-chloroform to afford a pure specimen as plates, mp 114°-15°identical with the natural product.

EXAMPLE 19 Combretastatin A-1

Method B. A solution of silyl either (10.78 g 19.26 mmol) intetrahydrofuran (100 mL under argon) was treated with tetrabutylammoniumfluoride (45 mL, 1M solution in tetrahydrofuran) and stirred for 10minutes. After completion the reaction mixture was extracted with ether(300 mL). The ethereal solution was washed with cold water (2×100 mL),dried and evaporated to a powder (1a, 6.0 g, 93.8%), which crystallizedfrown chloroform-hexane as plates, mp 113°-15°. Anal. calcd for C₁₈ H₂₀O₆, C, 65.06;H 6.07. Found: C, 64.48; H, 6.03.

EXAMPLE 20 Microtubule Assembly

The assembly reaction at 37° C. was followed turbidimetrically asdescribed by Hamel et al, Biochem. Pharmacal., 32, p. 3864, 1983; andBatra et al, Molecular Pharm. 27, pp 94-102, 1984. Each 0.25 ml reactionmixture contained 1.5 mg/ml of tubulin and 0.5 mg/ml ofmicrotubule-associated proteins (proteins were purified as described byHamel et al, Biochemistry, 23, p. 4173, 1984, 0.1M 4-morpholineethanesulfonate (adjusted to pH 6.6 with NaOH), 0.5 mM MgCl₂ 0.5 mMguanosine 5'-triphosphate, and drugs as required. The concentration ofdrug needed to inhibit the extent of assembly by 50% was determined.

EXAMPLE 21 Binding of [³ H]colchicine to tubulin

Binding of radiolabeled colchicine to tubulin was measured by retentionof drug-tubulin complex on DEAE-cellulose paper filters, as described byHamel et al, Biochem. Pharmacal., supra, Reaction mixtures (0.1 ml)contained 0.1 mg/ml of tubulin, 5 μM [³ H]colchicine, the competing drugat 5 μM 1.0M monosodium glutamate (adjusted to pH 6.6 with HCl), 0.1Mglucose-1-phosphate, 1 mM MgCl₂, 1 mM guanosine 5'-triphosphate, and 0.5mg/ml bovine serum albumin (the latter four components substantiallyenhance the rate of the reaction). Incubation was for 10 minutes at 37°C.

EXAMPLE 22 Acetylation of Combretastatin A-2 and A-3

Both combretastatin A-2 (12.0 mg) and combretastatin A-3 (18.0 mg) wereacetylated (separately) with acetic anhydride (1.0 ml)-pyridine (0.5 ml)at room temperature (72 hrs). Solvent was evaporated under a stream ofnitrogen to afford the acetate and diacetate as viscous oils:Combretastatin A-2 acetate displayed Rf. 0.60 (1:1 hexane-ethylacetate); IR υ_(max) (NaCl) 1767, 1510, 1430, 1264, 1199, 1127, 1112,1086, 1042, 930, 773 cm⁻¹ ; ¹ H-NMR (400 MHz) 2.273 (3H, s, COCH₃),3.731 (3H, s, OCH₃), 3.809 (3H, s, OCH₃), 5.940 (2H, s, --OCH₂ O--),6.410 (2H, s, --CH═CH--), 6.453 (1H, d, J=1.1 Hz, H-2, or H-6), 6.473(1H, d, J=1.1 Hz, H-6 or H-2), 6.840 (1H, d, J=8.56 Hz, H-5'), 6.977(1H, d, J=2.0 Hz, H-2'), 7.107 (1H, dd, J=8.56, 2.0 Hz, H-6'); HREIMS(m/z) 342.1101 (59, M⁺, calcd for C₁₉ H₁₈ O₆ : 342.1103), 300.0987 (100,C₁₇ H₁₆ O₅); and Combretastatin A-3 diacetate displayed Rf. 0.54 (1:1hexane-ethyl acetate); IR υ_(max) (NaCl) 1769, 1510, 1370, 1284, 1265,1242, 1203, 1132, 1113, 1092 cm⁻¹ ; ¹ H-NMR (400 MHz) 2.270 (3H, s,COCH₃), 2.287 (3H, s, COCH₃), 3.675 (3H, s, OCH₃), 3.807 (3H, s, OCH₃),3.813 (3H, s, OCH₃), 6.390 (1H, d, J=12.1 Hz, --CH═CH--), 6.445 (1H, d,J=12.2 Hz, --CH═CH--), 6.634 (1H, d, J=1.76 Hz, H-6), 6.708 (1H, d,J=1.76 Hz, H-2), 6.849 (1H, d, J=8.46 Hz, H-5'), 7.037 (1H, d, J=2.0 Hz,H-2'), 7.115 (1H, dd, J=8.46, 2.0 Hz, H-6'); HREIMS (m/z) 386.1365 (71,M⁺, calcd for C₂₁ H₂₂ O₇ : 386.1366), 344.1252 (56, C₁₉ H₂₀ O₆) 302.1146(100, C₁₇ H₁₈ O₅).

EXAMPLE 23 Hydrogenation of Combretastatin A-2 and Combretastatin A-3

In separate experiments combretastatin A-2 (12.0 mg) and combretastatinA-3 (10 mg) in methanol (10 ml ) and 5% Pd/c (10 mg) were each treatedwith a positive pressure of hydrogen at ambient temperature overnight.Catalyst was removed by filtering the solution and the product purifiedby preparative layer chromatography on regular (250μ) Analtex plateswith hexane-ethyl acetate (1:1) as mobile phase. The oily product fromcombretastatin A-3 was identical with natural combretastatin B-2 whilethe dihydro product i.e., bibenzyl, from combretastatin A-2 was aviscous oil exhibiting Rf. 0.58 (1:1, hexane-ethyl acetate); IR υ_(max)(NaCl) 3478, 1633, 1590, 1510, 1451, 1441, 1329, 1274, 1194, 1129, 1089,925 cm⁻¹ ; ¹ H-NMR (90 MHz) 2.78 (4H, s, --CH₂ CH₂ --), 3.86 (3H, s,OCH₃), 3.87 (3H, s, OCH₃), 5.93 (2H, s, --OCH₂ O--), 6.30 (H, d, J=1.5Hz, H-2 or H-6), 6.38 (1H, d, J=l.5 Hz, H-6 or H-2), 6.63 (1H, dd,J=8.2, 1.9 Hz, H-6'), 6.76 (1H, d, J=8.2 Hz, H-5'), 6.77 (1H, d, J=1.9Hz, H-2'); an HREIMS (m/z) 302.1149 (29, M⁺, calcd for C₁₇ H₁₈ O₅ :302.1154), 165.0547 (100, calcd for C₉ H₉ O₃ : 165.05521), 137.0597 (67,calcd for C₈ H₉ O₂ : 137.06031).

EXAMPLE 24 Combretastatin A-2/3'-O-p-bromophenylcarbamate

To a solution of combretastatin A-2 (10 mg) in dry methylene chloride (1ml) was added to a solution of p-bromophenyl isocyanate (80 mg) inmethylene chloride (1 ml). The mixture was stirred for 5 days at roomtemperature and heated at reflux for 24 hours. The reaction mixture wascooled to room temperature and the solution filtered. The filtrate wasconcentrated and chromatographed by preparative layer with hexane-ethylacetate (1:1) as mobile phase. The band was eluted with acetone andcrystallized from methylene chloride to afford an amorphous powder, mp138°-140°: IR υ_(max) (NaCl) 3310, 1734, 1722, 1533, 1509, 1491, 1431,1398, 1202, 1128, 1114, 924, 750 cm⁻¹ ; ¹ H-NMR (90 MHz ), 3.75 (3H, s,(OCH₃), 3.94 (3H, s, OCH₃), 5.93 (2H, s, OCH₂ O), 6.43 (2H, brs,--CH═CH--), 6.48 (2H, AB_(q), J=1.1 Hz, H-2,H-6), 6.65-7.0 (3H, M, H-2',5', 6'), 7.09 (1H, s, NH), 7.35 (2H, d, J=8.5 Hz, ArH ), 7.41 (2H, d,J=8.5 Hz, ArH). HRFAB: 500, 498.055218 (M⁺ +H, calcd for C₂₄ H₂₁ O₆NBr⁷⁹ : 498.058850).

EXAMPLE 25 Methyl-3,4-dihydroxy-5-methoxy-benzoate

Methyl gallate (10 g, 54.3 mmol) was added to a solution of borax (80 g)in 800 ml of water with stirring (30 min). Dimethyl sulfate (30 ml) andsolution of sodium hydroxide (13 g in 50 ml of water) were added fromtwo separate dropping funnels over 2.5 hours and stirring was continuedovernight. Concentrated sulfuric acid (50 ml) was added and stirringcontinued an additional hour. The product was extracted with chloroform(5×11 and each time stirring the solution for 20 minutes). The combinedchloroform extract was washed with brine (500 ml ), dried, concentratedand the residue crystallized from methanol-benzene to yield methyl ether(9.1 g, 84.5%): mp 110°-111° (lit. ¹⁹ mp 112°); IRυ_(max) (NaCl) 3380,1700, 1696, 1611, 1436, 1341, 1314, 1229, 1204, 1089 cm⁻¹ ; and ¹ H-NMR(90 MHz ), 3.88 (3H, s, OCH₃), 3.94 (3H, s, OCH₃), 5.50-6.0 (2H, OH),7.22 (1H, d, J=1.8 Hz, ArH), 7.34 (1H, d, J=1.8 Hz, ArH). Underanalogous experimental conditions but using continuous extraction withethyl acetate in place of chloroform, mixture of methyl gallate (7.0 g)and 3-0-methyl-gallic acid (1.4 g) was obtained.

EXAMPLE 26 Methyl 3,4-methylenedioxy-5-methoxy-benzoate

Cesium fluoride (24.5 g, 161.5 mmol) was added to a stirring solution ofthe phenol prepared in Example 25 (7.3 g, 36.8 mmol) indimethylformamide (90 ml) under argon. After stirring 20 minutes,dibromomethane (2.8 ml, 40.5 mmol) was added and the mixture heated for2 hours. The reaction was allowed to cool to room temperature. Ether(300 ml) was added and the ethereal solution was washed with cold water(3×50 ml), dried and concentrated to afford the methylenedioxyderivative as a powder (7.62 g, 98%) yield, which was recrystallizedfrom acetone-hexane, mp 89°-91°: IR υ_(max) (NaCl) 1714, 1636, 1507,1436, 1369, 1327, 1245, 1177, 1107, 1041 cm⁻¹ ; ¹ H-NMR (90 MHz) 3.89(3H, s, OCH₃), 6.06 (2H, s, --CH₂ --), 7.21 (1H, d, J-1.4 Hz, ArH), 7.33(1H, d, J= 1.4 Hz, ArH) and EIMS, m/z (rel. int/%) 210 (100, M⁺), 179,M⁺ -OCH₃).Anal. Calcd for C₁₀ H₁₀ O₅ : C, 57.15;H, 4.80 Found: C,57.10;H, 4.74.

EXAMPLE 27 3,4-Methylenedioxy-5-methoxy-benzyl alcohol

Lithium aluminum hydride (0.50 g) was added to a stirred solution of themethyl ester prepared in Example 26 (1.7 g) in ether-tetrahydrofuran(2:1, 50 ml). After stirring for 30 minutes, the reaction mixture wascooled to 5° C. and saturated aqueous sodium sulfate was carefully addeduntil a white solid appeared. The precipitate was collected byfiltration and the solution was dried and solvent evaporated to givecrystalline benzyl alcohol (1.45 g, 98% yield). Recrystallization fromethyl acetate-hexane afforded an analytical sample melting at 66°-67° C.(lit.²¹ mp 66): IR υ_(max) (NaCl) 3220, 1632, 1467, 1453, 1323, 1203,1133, 1093, 1008, 918 cm⁻¹ ; and ¹ H-NMR (90 MHz) 1.75 (1H, brs, OH),3.90 (3H, s, OCH₃), 4.58 (2H, brs, --CH₂ OH), 5.96 (2H, s, --CH₂ --),6.55 (2H, s, ArH) .

EXAMPLE 28 3,4-Methylenedioxy-5-methoxy-benzaldehyde

To a stirred yellow mixture of pyridinium chlorochromate (1.72 g, 7.99mmol) and anhydrous sodium acetate (0.655 g, 7.99 mmol) in CH₂ CL₂ (30ml) was added at once a solution of benzyl alcohol 7e (1.32 g, 7.26mmol) in CH₂ Cl₂ (10 mol). The greyish solution which formed immediatelywas stirred for 2 hour, and the reaction was monitored by TLC (1:1hexane-ethyl acetate). After filtering the solution through a smallsilica column colorless aldehyde (1.18 g, 91% yield) was eluted withhexane-ethyl acetate (7:3). Recrystallization from acetone affordedneedles, mp 132°-133° C. (lit. mp 130-31, 129-30): IR υ_(max) (NaCl)1694, 1676, 1622, 1508, 1474, 1451, 1362, 1325, 1134, 1190 cm¹ ; and ¹H-NMR (90 MHz), 3.95 (3H, s, OCH₃), 6.09 (2H, s, --CH₂ --), 7.04 (1H, d,J=1.4 Hz, ArH), 7.12 (1H, d, J=1.4 Hz, ArH), 9.78 (1H, s, --CHO).

EXAMPLE 29 3-Hydroxy-4-methoxy-benzyl-triphenylphosphonium/bromide

A solution of phosphorus tribromide (8.51 ml) in a mixture oftetrahydrofuran-benzene (1:2, 330 ml) was added to a cool (0° C.)solution of 3-hydroxy-4-methoxy-benzyl alcohol (6.13 g, 40 mmol) in thesame solvent (75 ml) under argon. The colorless solution was stirred atroom temperature for 2 hours, poured onto ice water (100 ml) andextracted with ether (2×100 ml). The ethereal layer was washed once withwater (50 ml), followed by brine (2×50 ml), dried and evaporated todryness to afford the bromide as an amorphous powder. A solutionprepared from the crude bromide, anhydrous-benzene (150 ml) andtriphenylphosphine (15.72 g, 60 mmol) was stirred for 10 minutes at roomtemperature and heated at reflux for 2 hours. On cooling to roomtemperature, a viscous oil separated. The upper solvent phase wasdecanted and the oil was crystallized from ethanol-ether to give thebromide as a powder (10.0 g, 52.4% from alcohol ): mp 262°-4° C.; IRυ_(max) (NaCl) 3158, 1604, 1589, 1527, 1512, 1437, 1279, 1255, 1128,1111, 743 cm⁻¹ ; and ¹ H-NMR (90 MHz, CDCl₃ +D₂ O), 3.77 (3H, s, OCH3),4.95 (2H, d, J_(PCCH) =13.7 Hz, --CH₂ --), 6.57 (2H, brs, ArH)), 6.83(1H, brs, ArH), 7.56-7.77 (15H, ArH). Anal. Calcd for C₂₆ H₂₄ O₂ PB_(r): C, 65.6;H, 5.05; Br, 16.67. Found C, 64.50; H, 5.07; Br, 16.78.

EXAMPLE 30 1-Hydroxy-3,4-methylenedioxy-4',5-dimethoxy-(E)-and(Z)-stilbene, Combretastatin A-2 and E-isomer

Phosphonium bromide (3.35 g, 7.0 mmol) was suspended in tetrahydrofuran(100 ml), stirred, cooled to -50° C. (under argon), and n-butyllithium(10 ml, 15 mmol) was added using a syringe and septum technique. Thesolution became deep red immediately and upon reaching room temperaturewas stirred 20 minutes prior to adding a solution of the aldehydeobtained from Example 28 (1.15 g, 6.39 mmol) in tetrahydrofuran (30 ml).All the aldehyde was consumed in 30 minutes. Cold hydrochloric acid (1N,50 ml)was added followed by water (100 ml), and the product wasextracted with ethyl acetate (3×100 ml) from the colorless solution. Theethyl acetate extract was washed with water (50 ml), brine (50 ml),dried and solvent evaporated. The crude product was chromatographed on asilica gel (50 g) column. Elution with hexane-ethyl acetate (17:3)afforded a mixture of Z and E stilbenes (1.09 g, 57% yield, ratio Z/E,1:16). Half of the product was rechromatographed on a longer silica gelcolumn and elution with hexane-ethyl acetate (9:1) provided firstcombretastatin A-2 (40 mg ) as a viscous oil identical with naturalcombretastatin A-2 and later the E-isomer (0.20 g) as needles from ethylacetate-hexane, mp 145°-50° C.: IR υ_(max) (NaCl) 3490, 1622, 11509,1463, 1440, 1429, 1319, 1279, 1263, 1254, 1133 cm⁻¹ ; and ¹ H-NMR (400MHz) 3.908 (3H, s, OCH₃), 3.941 (3H, s, OCH₃), 5.598 (1H, brs, OH),5.978 (2H, s, --OCH₂ O--), 6.629 (1H, d, J=1.30 Hz, H-2 or H-6), 6.729(1H, d, J=1.30 Hz, H-6 or H-2), 6.825 (1H, d, J=8.32 Hz, H-5'), 6.848(2H, s, --CH═CH--), 6.945 (1H, d, J=8.32, 2.0 Hz, H-6'), 7.113 (1H, d,J=2.0 Hz, H-2').

Anal. Calcd for C₁₇ H₁₆ O₅ : C, 68.00;H, 5.37. Found C, 67.63;H, 5.37.

EXAMPLE 31 Photochemical isomerization of E-stilbene to combretastatinA-2

A solution of E-isomer (40 mg) in dioxane (30 ml) and water (1 ml)wasstirred and irradiated (directly into the solution from above) with longwave (365 nm) length UV for 5 hours. The ultraviolet source was UV lampused for visualizing TLC plates equipped with both short-wave (254 nm)and long wave (365 nm) lamps. The solvent was removed and ¹ H-NMRexamination of the residue revealed an isomeric mixture in the ratio Z/Eof 2.5:1.5. Separation by chromatography on a silica gel column andelution with hexane-ethyl acetate (9:1) yielded combretastatin A-2 (15mg).

EXAMPLE 32tert-Butyldimethylsilyl)3-[(tert-Butyldimethylsilyl)-oxy]-4,5,-dimethoxy-benzoate

Diisopropylethylamine (11.3 ml, 77 mmol) was added to a stirred solutionof 3-hydroxy-4,5-dimethoxy-benzoic acid (5.0 g, 25 mmol) indimethylformamide (50 ml, under argon) followed by addition oftert-butyldimethylsilyl chloride (8.32 g, 55 mmol) and the reactionmixture was stirred for 1 hour. Ice (50 g) was added and the reactionmixture was extracted with ethyl ether (200 ml). The ethereal solutionwas washed with cold water (3×50 ml), sodium bicarbonate solution (10%,50 ml), water (50 ml), dried and solvent evaporated to yieldtert-butyldimethylsilyl3-[(tert-Butyldimethylsilyl)-oxy]-4,5,-dimethoxy-benzoate as achromatographically homogeneous oil (10.4 g, 97% yield). Attempts athigh vacuum distillation (100° at 1.0 mm/Hg) failed and resulted indesilylation. However, the oil showed the correct IR and ¹ H-NMRspectral data for the silane: IR υ_(max) (NaCl) 2932, 1700, 1590, 1420,1350, 1254, 1230, 1221, 1118, 839, 775 cm⁻¹ and ¹ H-NMR (90 MHz) 0.107(6H, s, 2×SiCH₃), 0.276 (6H, s, 2×SiCH₃), 0.922 (18H, s, 6 ×CH₃), 3.751(3H, s, OCH₃), 3.790 (3H, s, OCH₃), 7.170 (2H, AB_(q), J_(AB) =1.5 Hz,ArH) .

EXAMPLE 33 3-[(tert-Butyldimethylsilyl)-oxy]-4,5,-dimethoxy-benzylalcohol

The silyl ester (10.0 g, 23 mmol)was dissolved in ether (300 ml) andstirred (under argon) with lithium aluminum hydride (2.0 g) at roomtemperature for 1 hour. Saturated ammonium chloride solution (ice-cold)was added and the ether layer separated. The aqueous phase was extractedwith ether (3×200 ml) and the combined ether extract was washed withsodium bicarbonate solution, cold water, and dried. After solventremoval, the residual oil was found to be chromatographicallyhomogeneous alcohol (6.0 g, 86% yield): the oil distilled at 210° C.(0.04 mm) and displayed IR υ_(max) (NaCl) 3450, 2931, 1587, 1501, 1427,1233, 1118, 1004, 837, 782 cm⁻¹ ; and ¹ H-NMR (90 MHz) 0.201 (6H, s,2×CH₃), 1.026 (9H, s, 3×CH₃), 1.716 (1H, t, J=5.9 Hz, OH D₂ O exchanged)3.794 (3H, s, OCH₃), 4.596 (2H, d, J=5.9 Hz, --CH₂ OH, collapsed to abroad singlet upon D₂ O exchange), 6.510 (1H, d, J=1.9 Hz, ArH), 6.610(1H, d, J=1.9 Hz, ArH). Anal. Calcd for C₁₆ H₂₆ O₄ Si: C, 60.39, H, 8.78Found C, 60.06;H, 8.78.

EXAMPLE 343-[(tert-Butyldimethylsilyl)-oxy]-4,5-dimethoxy-benzyl-bromide

Before adding phosphorus tribromide (0.95 ml) in methylene chloride (5ml) a solution of the silyloxy-benzyl alcohol prepared in Example 33(6.0, 20 mmol) in methylene chloride (anhydrous, 100 ml) was stirred andcooled (-10°, ice-salt bath) for 15 minutes. The mixture was stirred 10minutes and 10% aqueous sodium bicarbonate solution (50 ml) was added(slowly). The methylene chloride layer was washed with cold water (2×50ml), dried and solvent evaporated to give3-[(tert-butyldimethylsilyl)-oxy]-4,5-dimethoxy-benzyl-bromide as acolorless oil (6.4 g, 88% yield), homogeneous by TLC. While the productthus formed was heat sensitive, and distillation was unsuccessful, itdid give the correct IR υ_(max) (NaCl) 2932, 1586, 1500, 1427, 1348,1250, 1234, 1127, 1111, 838 cm⁻¹ ; ¹ H-NMR (90 MHz), 0.183 (6H, s,2×CH₃), 1.006 (9H, s, 3×CH₃), 3.777 (3H, s, OCH₃), 3.855 (3H, s, OCH₃),4.409 (2H, s, --CH₂ --), 6.561 (2H, AB_(q), J=2.0 Hz, ArH); and MS (m/z,rel. amt.) 362, 360 (50%, M⁺) 305, 303, (80, M⁺ -C₄ H₉), 281 (60, M⁺-Br), 253 (75, M⁺ -Br-28), 209 (100, M⁺ -Br-C₅ H₁₂), 166 (45, M⁺-Br-Si(CH₃)₂ C (CH₃)3].

EXAMPLE 353-[(tert-Butyldimethylsilyl)-oxy]-4,5-dimethoxy-benzyl-tri-phenylphosphonium-bromide

To a solution of the bromide prepared in Example 34 (6.0 g, 16.6 mmol)in toluene (50 ml) was added (stirring) a solution of triphenylphosphine(4.36 g, 16.6 mmol) in toluene (10 ml). The mixture was heated to refluxfor 15 minutes. When the clear solution started to become turbid,heating was discontinued and the mixture was stirred overnight at roomtemperature. The solid phosphonium bromide (7.17 g, 69% yield) wasrecovered by filtration as a powder melting at 248°; IR υ_(max) (NaCl)2957, 1586, 1502, 1453, 1436, 1344, 1253, 1113, 836, 742, 721 cm⁻¹ and ¹H-NMR (90 MHz) 0.58 (6H, s, 2×CH₃), 1.48 (9H, s, 3×CH₃), 4.15 (3H, s,OCH₃), 4.32 (3H, s, OCH₃), 5.89 (2H, d, J_(PCH) =14 Hz, CH₂), 6.71 (1H,t, J=2.2 Hz, ArH), 7.36 (1 H, t, J=2.2 Hz, ArH), 8.23-8.45 (15H, ArH).Anal. Calcd for C₃₃ H₄₀ BrO₃ PSi: C, 63.56;H, 6.46; Br, 12.81. Found: C,64.04;H, 6.57; Br, 12.47.

EXAMPLE 36 Silyl-Combretastatin A-3

Butyllithium (2.47 ml, 2.2 mmol) was added to a stirred and cooled (-10°C.) suspension of phosphonium bromide (1.31 g, 2.1 mmol) intetrahydrofuran (100 ml). The orange-red solution was stirred at roomtemperature 10 minutes. 3-[(tert-butyldimethylsilyl)-oxy]-4,methoxy-benzaldehyde (0.532 g, 2.0 mmol) was added and stirringcontinued another 10 minutes while the red solution changed to yellow. ATLC examination (4:1, hexane-ethyl acetate) showed completion ofreaction. Ice water (100 ml) was added and the product extracted withether (3×100 ml ). The ethereal solution was washed with water (100 ml ), and concentrated to a gum which upon silica gel column (40 g)chromatography and elution with hexane-ethyl acetate (97:3) afforded amixture of 3,3'-bis-[(tert-butyldimethylsilyl)-oxy], 4',4,5-trimethoxy-(Z)- and (E)-stilbene in a ratio of 5:1 (0.870 g, 82%yield). The isomers were separated by preparative layer chromatographyon silica gel (20×20 cm, 500 m, E-Merck plates) employing 19:1hexane-ethyl acetate. The short UV positive upper band was major productand elution with hexane-ethyl acetate yielded Z-isomer as an oil (423mg): IR υ_(max) (NaCl) 2930, 1575, 1509, 1250, 1231, 1118, 838, 782 cm⁻¹and ¹ H-NMR (400 MHz) 0.070 (6H, s, 2×CH₃), 0.105 (6H, s, 2×CH₃), 0.932(9H, s, 3×CH₃), 0.958 (9H, s, 3×CH₃), 3.666 (3H, s, OCH₃), 3.761 (3H, s,OCH₃), 3.774 (3H, s, OCH₃), 6.366 (1H, d, J=12 Hz, --CH═CH--), 6.413(1H, d, J=1.84 Hz, H-6), 6.429 (1H, d, J=12 Hz, --CH═CH--), 6.471 (1H,d, J=1.84 Hz, H-2), 6.718 (1H, dd, J=8.3 Hz, H-5'), 6.778 (1H, d, J=2.0Hz, H-2'), 6.840 (1H, dd, J=8.3, 2.0 Hz, H-6 '). The lower long UVpositive band was also eluted with hexane-ethyl acetate to give anE-isomer as an oil (80 mg): IR υ_(max) (NaCl) 2930, 1578, 1509, 1427,1272, 1251, 1117, 838, 782 cm⁻¹ and ¹ H-NMR (400 MHz) 0.183 (6H, s,2×CH₃), 0.207 (6H, s, 2×CH₃), 1.024 (9H, s, 3×CH₃), 1.028 (9H, s,3×CH₃), 3.794 (3H, s, OCH₃), 3.823 (3H, s, OCH₃), 3.901 (3H, s, OCH₃),6.626 (1H, d, J=1.88 Hz, H-6), 6.693 (1H, d, J=1.88 Hz, H-2), 6.800 (1H,d, J=16.2 Hz, --CH═CH--), 6.825 (1H, d, J=8.3 Hz, H-5'), 6.845 (1H, d,J=16.2 Hz, --CH═CH--), 7.020 (1H, d, J=2.100,H-2'), 7.047 (1H, dd, J=8.3Hz, 2.1 Hz, H-6'). Anal. Calcd for C₂₉ H₄₆ O₅ Si₂ : C, 65.62;H, 8.73.Found C, 66.09;H, 8.97.

EXAMPLE 37 Combretastatin A-3

To a stirred solution of silyl-Z-stilbene (0.25 g, 0.47 mmol) intetrahydrofuran (10 ml under argon) was added a 1M tetrahydrofuransolution of tetrabutylammonium fluoride (1 ml, 1.0 mmol).Instantaneously the solution became yellow and reaction was complete asevidenced by TLC (hexane-ethyl acetate 3:2). Ice (5 g) and water (5 ml)was added to the mixture and the product extracted with ether (2×25 ml).The ethanol extract was washed with cold water (20 ml), and dried. Afterevaporation of solvent the residue in 1:1 hexane-ethyl acetate wasfiltered through a pipette filled with silica gel (2 g) to affordcombretastatin A-3 as an oil (0.13 g, 91% yield), homogeneous by TLC andidentical with the natural product.

EXAMPLE 38 Methylation of Combretastatins B-2 and B-3

Combretastatin B-2 (10mg) and combretastatin B-3 (2.0 mg) wereseparately methylated in a refluxing (5 hr) mixture composed of excessmethyl iodide, potassium carbonate and acetone. The potassium carbonatewas collected by filtration and the permethyl ether derivative wasisolated by passing the filtrate through a pipette filled with silicagel. The products from both reactions were found to be identical viscousoils: IR√υ_(max) 1589 1514 1509 1464 1457 1419 1261 1236, 1127 cm⁻¹ ; ¹H-NMR (400 MHz) 2.849 (4H, m, ARCH₂), 3.825 (H, s, OCH₃), 3.844 (3H, s,OCH₃), 3.863 (3H, s, OCH₃), 6.368 (2H, s, ArH), 6,662 (1H, d, J=1.88 Hz,ArH), 6.724 (1H, dd, J=8.10, 1.88 Hz, ArH), 6.802 (1H, d, J=8.10 Hz,ArH); HREIMS (m/z) 332.1621 (M⁺, 18%, calcd for C.sub. 19 H₂₄ O₅ :332.1624), 181.0864 (100%, calcd for C₁₀ H₁₃ O₃ : 181.0865), 151,0762(50%, calcd for C₉ H₁₁ O₂ : 151.0759).

EXAMPLE 39 Combretastatin B-4 Permethyl ether

Combretastatin B-4 (20 mg) and the 3'-hydroxy-3,4', 5-trimethoxybibenzyl (6.2 mg) were each permethylated employing excess methyl iodideand potassium carbonate in acetone as described above to give identicalproducts: permethyl ether as viscous oils: IR υ_(max) 1606, 1594, 1514,1463, 1428, 1419, 1204, 1151 cm⁻¹ ; ¹ H-NMR (400 MHz) 2.843 (4H, s,ArCH₂), 3.763 (6H, s, 2×OCH₃), 3.843 (3H, s, OCH₃), 3.857 (3H, s, OCH₃),6.311 (1H, t, J=2.4 Hz, ArH), 6.338 (2H, d, J=2.4 Hz, ArH), 6.672 (1H,d, J=1.9 Hz, ArH), 6.728 (1H, dd, J=8.2, 1.9 Hz, ArH), 6.792 (1H, d,J=1.9 Hz, ArH); and HREIMS (m/z) 302.1511 (M⁺, 14%, calcd for C₁₈ H₂, O₄: 302.1518), 151.0761 (100%, calcd for C₉ H₁₁ O₂ : 151.0759).

EXAMPLE 40 3,4-Dibenzyloxy-benzaldehyde

A mixture of 3,4-dihydroxy-benzaldehyde (2.76 g, 20 mmol) in dry acetone(50 ml), potassium carbonate (5.52 g, 40 mmol) and benzylbromide (5 ml,42 mmol) was heated at reflux for 12 hours. The mixture was cooled toroom temperature, potassium carbonate was removed by filtering thesolution and the filtrate was concentrated to a powder which wasrecrystallized from acetone to yield (5.4 g, 81%) ether as prisms, mp89°-90° (lit. mp 90°) IR υ_(max) 1685, 1595, 1483, 1508, 1454, 1434,1269, 1132, 695, 650 cm⁻¹ ; and ¹ H-NMR (90 MHz), 5.21 (2H, s, ArCH₂ O),5.26 (2H, s, ArCH₂₀), 7.02 (1H, d, J=7.9 Hz), 7.30-7.50 (12H, ArH), 9.81(1H, s, CHO). Anal. calcd for C₂₁ H₁₈ O₃ : C, 79.23; H, 5.70. Found: C,79.26;H, 5.68.

EXAMPLE 41 3',4'-Dibenzyloxy-3,4,5-trimethoxy-(Z)-and (E)-stilbene

To a stirred suspension of sodium hydride (0.75, 31.4 mmol) inN,N-dimethylimidazolidinone (10 ml) was added3,4,5-trimethoxy-benzyl-phosphonium bromide (8.26 g 15.71 mmol) underargon. The aldehyde (4.0 g, 1258 mmol) from Example 40 was added to thedeep red solution followed by 2 ml of N,N-dimethylimidazolidinone.Before adding ice (10 g)-water (75 ml) the mixture was stirredovernight. Upon extraction with ethyl acetate (3×100 ml), the organicphase was washed with water (3×75 ml), dried and evaporated to a darkcolored mass (9.0 g). The crude product in hexane-ethyl acetate (9:1)was chromatographed on a column of silica gel (200 g) to afford amixture of E- and Z-isomers (3.70 g, 61% yield). A 1.1 g sample of themixture was rechromatographed on a column of silica gel and eluted withhexane-ethyl acetate (19:1) to furnish Z-isomer (0.25 g) and E-isomer(0.32 g). The Z-isomer was further purified by preparative TLC(hexane-ethyl acetate, 7:3) to yield3',4'-Dibenzyloxy-3,4,5-trimethoxy-(Z)-stilbene as a chromatographicallyhomogeneous and viscous oil: IR υ_(max) 1581, 1509, 1462, 1454, 1412,1265, 1237, 1128, 1007 cm⁻¹ ; ¹ H-NMR (400 MHz) 3.731 (6H, s, 2×OCH₃),3.856 (3H, s, OCH₃), 4.856 (2H, s, ArCH₂), 5.121 (2H, s, ArCH₂), 6.589(2H, s, ArH) , 6.600 (3H, m, ArH), 6.747 (1H, d, J=8.6 Hz, ArH), 6.758(1H, brs, ARH), 7.273-7.407 (10H, ArH).

The E-isomer crystallized from acetone-methanol as granules melting at1005°-106°: IR υ_(max) 1581, 1509, 1454, 1413, 1241, 1128, 1008 cm⁻¹ ;and ¹ H-nmr (400 MHz) 3.874 (3H, s, OCH₃), 3.909 (6H, s, 2×OCH₃), 5.219(4H, s, 2×ARCH₂), 6.855 (2H, s, ArH), 6.942 (1H, d, J=8.4 Hz, ArH),7.209 (1H, dd, J=-8.4, 2.2 Hz, ArH), 7.261 (2H, s, --CH═CH--), 7.318(1H, d, J=2.2 Hz, ArH), 7.325-7.500 (10H, m, ArH).

EXAMPLE 42 Combretastatin B-3

A mixture composed of Z- and E-isomers (0.35 g), 5% pd/C (0.10 g) andmethanol-ethyl acetate (1:1, 20 ml) was saturated (ambient temperature )with hydrogen at a slightly positive pressure. The reaction mixture wasstirred overnight, catalyst was removed by filtration and the crudeproduct was chromatographed (silica gel column). Elution withhexane-ethyl acetate (4:1) yielded combretastatin B-3 (0.20 g, 91%) as asticky oil which crystallized from ethanol-ether as rods: mp 114°-16°and identical with the natural product.

EXAMPLE 43 Dosage Forms

Several dosage forms were prepared embodying the present invention. Theyare shown in the following examples in which the notation "activeingredient" signifies one of the disclosed combretastatins, namely, A-1,A-2, A-3, B-1, B-2, B-3 or B-4, their synthetic counterparts and thenon-toxic pharmaceutically active derivatives thereof.

COMPOSITION "A" Hard-Gelatin Capsules

One thousand two-piece hard gelation capsules for oral use, each;capsule containing 200 mg of an active ingredient are prepared from thefollowing types and amounts of ingredients:

    ______________________________________                                        Active ingredient, micronized                                                                         200    gm                                             Corn Starch             20     gm                                             Talc                    20     gm                                             Magnesium stearate      2      gm                                             ______________________________________                                    

The active ingredient, finely divided by means of an air micronizer, isadded to the other finely powdered ingredients, mixed thoroughly andthen encapsulated in the usual manner.

The foregoing capsules are useful for treating a neoplastic disease bythe oral administration of one or two capsules one to four times a day.

Using the procedure above, capsules are similarly prepared containing aactive ingredient in 50, 250 and 500 mg amounts by substituting 50 gm,250 gm and 500 gm of a active ingredient for the 200 gm used above.

COMPOSITION "B" Soft Gelatin Capsules

One-piece soft gelatin capsules for oral use, each containing 200 mg ofa active ingredient (finely divided by means of an air micronizer), areprepared by first suspending the compound in 0.5 ml of corn oil torender the material capsulatable and then capsulating in the abovemanner.

The foregoing capsules are useful for treating a neoplastic disease bythe oral administration of one or two capsules one to four times a day.

COMPOSITION "C" Tablets

One thousand tablets, each containing 200 mg of a active ingredient areprepared from the following types and amounts of ingredients:

    ______________________________________                                        Active ingredient micronized                                                                           200 gm                                               Lactose                  300 gm                                               Corn starch              50 gm                                                Magnesium stearate       4 gm                                                 Light liquid petrolatum  5 gm                                                 ______________________________________                                    

The active ingredient finely divided by means of an air micronizer, isadded to the other ingredients and then thoroughly mixed and slugged.The slugs are broken down by forcing through a Number Sixteen screen.The resulting granules are then compressed into tablets, each tabletcontaining 200 mg of the active ingredient.

The foregoing tablets are useful for treating a neoplastic disease bythe oral administration of one or two tablets one to four times a day.

Using the procedure above, tablets are similarly prepared containing aactive ingredient in 250 mg and 100 mg amounts by substituting 250 gmand 100 gm of a active ingredient for the 200 gm used above.

COMPOSITION "D" Oral Suspension

One thousand ml of an aqueous suspension for oral use, containing ineach teaspoonful (5 ml) dose, 50 mg, of a active ingredient, is preparedfrom the following types and amounts of ingredients:

    ______________________________________                                        Active ingredient micronized                                                                           10 gm                                                Citric acid              2 gm                                                 Benzoic acid             1 gm                                                 Sucrose                  790 gm                                               Tragacanth               5 gm                                                 Lemon Oil                2 gm                                                 Deionized water, q.s. 1000 ml.                                                ______________________________________                                    

The citric acid, benzoic acid, sucrose, tragacanth and lemon oil aredispersed in sufficient water to make 850 ml of suspension. The activeingredient finely divided by means of an air micronizer, is stirred intothe syrup until uniformly distributed. Sufficient water is added to make1000 mi.

The composition so prepared is useful for treating a neoplastic diseaseat a dose of 1 tablespoonful (15 ml) three times a day.

COMPOSITION "E" Parenteral Product

A sterile aqueous suspension for parenteral injection, containing in 1ml 300 mg of a active ingredient for treating a neoplastic disease, isprepared from the following types and amounts of ingredients:

    ______________________________________                                        Active ingredient, micronized                                                                          30 gm                                                Plysorbate 80            5 gm                                                 Methylparaben            2.5 gm                                               Propylparaben            0.17 gm                                              Water for injection, q.s. 1000 ml.                                            ______________________________________                                    

All the ingredients, except the active ingredient, are dissolved in thewater and the solution sterilized by filtration. To the sterile solutionis added the sterilized active ingredient, finely divided by means of anair micronizer, and the final suspension is filled into sterile vialsand the vials sealed.

The composition so prepared is useful for treating a neoplastic diseaseat a dose of 1 milliliter (1 M) three times a day.

COMPOSITION "F" Suppository, Rectal and Vaginal

One thousand suppositories, each weighing 2.5 gm and containing 200 mgof a active ingredient are prepared from the following types and amountsof ingredients:

    ______________________________________                                        Active ingredient, micronized                                                                         15 gm                                                 Propylene glycol        150 gm                                                Polyethylene glycol #4000, q.s.                                                                       2,500 gm                                              ______________________________________                                    

The active ingredient is finely divided by means of an air micronizerand added to the propylene glycol and the mixture passed through acolloid mill until uniformly dispersed. The polyethylene glycol ismelted and the propylene glycol dispersion added slowly with stirring.The suspension is poured into unchilled molds at 40° C. The compositionis allowed to cool and solidify and then removed from the mold and eachsuppository foil wrapped.

The foregoing suppositories are inserted rectally or vaginally fortreating a neoplastic disease.

COMPOSITION "G" Intranasal Suspension

One thousand ml of a sterile aqueous suspension for intranasalinstillation, containing in each ml 200 mg of a active ingredient, isprepared from the following types and amounts of ingredients:

    ______________________________________                                        Active ingredient, micronized                                                                         15 gm                                                 Polysorbate 80          5 gm                                                  Methylparaben           2.5 gm                                                Propylparaben           0.17 gm                                               Deionized water, q.s. 1000 ml.                                                ______________________________________                                    

All the ingredients, except the active ingredient, are dissolved in thewater and the solution sterilized by filtration. To the sterile solutionis added the sterilized active ingredient, finely divided by means of anair micronizer, and the final suspension is aseptically filed intosterile containers.

The composition so prepared is useful for treating a neoplastic disease,by intranasal instillation of 0.2 to 0.5 ml given one to four times perday.

An active ingredient can also be present, as shown in Compositions H, I,and J in the undiluted pure form for use locally about the curls,intranasally, pharyngolaryngeally, bronchially, broncholially or orally.

COMPOSITION "H" Powder

Five grams of a active ingredient in bulk form is finely divided bymeans of an air micronizer. The micronized powder is placed in ashaker-type container.

The foregoing composition is useful for treating a neoplastic disease,at localized sites by applying a powder one to four times per day.

COMPOSITION "I" Oral Powder

One hundred grams of a active ingredient in bulk form is finely dividedby means of an air micronizer, The micronized powder is divided intoindividual doses of 200 mg and packaged.

The foregoing powders are useful for treating a neoplastic disease, bythe oral administration of one or two powders suspended in a glass ofwater, one to four times per day.

COMPOSITION "J" Insufflation

One hundred grams of a active ingredient in bulk form is finely dividedby means of an air micronizer.

The foregoing composition is useful for treating a neoplastic disease,by the inhalation of 300 mg one to four times per day.

COMPOSITION "K" Hard Gelatin Capsules

One hundred two-piece hard gelatin capsules for oral use, each capsulecontaining 200 mg of a active ingredient.

The active ingredient is finely divided by means of an air micronizerand encapsulated in the usual manner.

The foregoing capsules are useful for treating a neoplastic disease, bythe oral administration of one or two capsules, one to four times a day.

Using the procedure above, capsules are similarly prepared containingactive ingredient in 50, 250 and 500 mg amounts by substituting 50 gm,250 gm and 500 gm of the active ingredient for the 200 gm used above.

From the foregoing, it is apparent that an invention has been hereindescribed and illustrated which fulfills all of the aforestatedobjectives in a remarkably unexpected fashion. It is of courseunderstood that such modifications, alterations and adaptations as mayreadily occur to the art is an confronted with this disclosure areintended within the spirit of this disclosure which is limited only bythe scope of the claims appended hereto.

Accordingly, what is claimed is:
 1. The method of treating a mammalianhost afflicted with lymphocytic leukemia comprising administering tosaid host a pharmaceutical preparation containing as its essentialactive ingredient an effective amount of a substance having thestructural formula of either: ##STR6## wherein: R₁ is OH or OCH₃ and R₂is H or OCH₃ or R₁ R₂ is --OCH₂ O--, R₃ is H or OH, and R₄ is OH orOCH₃, with the proviso that when structure (I) is selected and R₁ isOCH₃, R₂ is OCH₃, R₃ is OH and R₄ is OCH₃ ; and that when structure (I)is selected and R₁ R₂ is --OCH₂ O--, R₃ is H and R₄ is OCH₃ ; and thatwhen structure (I) is selected and R₁ is OH, R₂ is OCH₃, R₃ is H and R₄is OCH₃ ; and that when structure (II) is selected and R₁ is OCH₃ and R₂is H or OH, R₃ is H and R₄ is OH; and that when structure (II) isselected and R₁ ═R₂ ═OCH.sub. 3, R₃ is OH and R₄ is OCH₃ ; and that whenstructure (II) is selected and R₁ is OH, R₂ is OCH₃, R₃ is H and R₄ isOCH₃.
 2. A method according to claim 1 in which said substance isdenominated combretastatin A-1 and has structural formula I wherein: R₁is OCH₃ ; R₂ is OCH₃ ; R₃ is OH and R₄ is OCH₃.
 3. A method according toclaim 1 in which said substance is denominated combretastatin A-2 andhas structural formula I wherein: R₁ R₂ is --OCH₂ O--; R₃ is H and R₄ isOCH₃.
 4. A method according to claim 1 in which said substance isdenominated combretastatin A-3 and has structural formula I wherein: R₁is OH; R₂ is OCH₃ ; R₃ is H and R₄ is OCH₃.
 5. A method according toclaim 1 in which said substance is denominated combretastatin B-1 andhas structural formula II wherein: R₁ is OCH₃ ; R₂ is OCH₃ ; R₃ is OHand R₄ is OCH₃.
 6. A method according to claim 1 in which said substanceis denominated combretastatin B-2 and has structural formula II wherein:R₁ is OH; R₂ is OCH₃ ; R₃ is H and R₄ is OCH₃.
 7. A method according toclaim 1 in which said substance has structural formula II wherein R₁ isOCH₃ ; R₂ is OCH₃ ; R₃ is H and R₄ is OH and is denominatedcombretastatin B-3 when R₂ is OCH₃ and is denominated combretastatin B-4when R₂ is H.